CN1103781C

CN1103781C - Abundant extracellular products and methods for production and use of same

Info

Publication number: CN1103781C
Application number: CN94194859A
Authority: CN
Inventors: M.A.霍维茨
Original assignee: University of California
Current assignee: University of California
Priority date: 1993-11-23
Filing date: 1994-11-18
Publication date: 2003-03-26
Anticipated expiration: 2014-11-18
Also published as: CA2177249A1; JPH09505588A; JP2005104983A; WO1995014713A2; CN1142231A; DE69435138D1; DK0771322T3; EP0771322B9; JP4537178B2; US6761894B1; ES2313719T3; AU1097795A; EP0771322B1; CA2177249C; BR9408135A; ATE407945T1; WO1995014713A3; EP0771322A1

Abstract

Vaccines based on combinations of majorly abundant extracellular products of pathogens and methods for their use and production are presented. The most prevalent or majorly abundant extracellular products of a target pathogen are selected irrespective of their absolute molecular immunogenicity and used as vaccines to stimulate a protective immune response in mammalian hosts against subsequent infection by the target pathogen. The majorly abundant extracellular products may be characterized and distinghuished by their respective N-terminal amino acid sequences. As the vaccines may comprise different combinations of the extracellular products, a broad range effective immunotherapeutic compositions are provided by the present invention. In addition to other infectious agents, the vaccines so produced can be used to stimulate an effective immune response against intracellular pathogens and in particular Mycobacterium tuberculosis.

Description

Abundant extracellular products and its production and using method

Cross-reference with related application

The application is that the sequence number submitted on November 23rd, 1993 is the part continuation application of 156,358 common unsettled U.S. Patent application, quotes the former as a reference at this. Relation with government

Fa Benming finishes under government supports, the Ministry of Health (the Department ofHealth and Human Services) give with grant number be A1-31338.Government enjoys some right to the present invention.

FIELD OF THE INVENTION

The present invention generally relates to the vaccine of immunotherapeutic agent and disease-resistant originality organism such as bacterium, protozoon, virus and fungi.In particular, different with the existing technology vaccine and the immunotherapeutic agent of pathogenic agent subunit that maximum or special molecular immunogenicity are arranged based on performance or product, the present invention uses the most general or main abundant immunogenic determinant that is discharged by pathogenic agent of selecting such as mycobacterium tuberculosis (Mycobat-terium tuberculosis) to stimulate the intravital effective immune response of mammalian hosts.Therefore, acquired immunity that produces by the present invention and immune therapeutic activity are to represent antigen sign on infected host cell at those the most normal during the pathogenic infection, and consider being correlated with or absolute immunity originality of the compound that come into operation not especially.

The background of invention

Recognize promptly that very early parasitic microbe has infection animal thereby causes that disease also usually causes the ability of host's death.Virulence factor is main causes of death always on whole history and continues to make the people to suffer huge misery.Though in the last hundred years in prevention with treat in many transmissible diseases and make remarkable progress, complicated host-parasite still universal validity of limit treatment means that interacts.Various diseases such as tuberculosis recur, and the appearance of many bacteriums and viral drug resistance strain has proved in antagonism by the difficulty in the meticulous infection mechanism of many pathogenicity bo carriers displaying.

In the related main pathogen factor of epidemiology, proved that bacterium is reluctant especially in the cell in face of treatment or preventive means.Comprise in the cell of Mycobacterium and legionella bacterium in the cell of infected host organisms rather than the extracellular finish its all or part of life cycle.Worldwide, there is every year the millions of people to die from intracellular bacterial infection and suffers huge misery.The tuberculosis that is caused by mycobacterium tuberculosis is whole world transmissible disease main causes of death, 10,000,000 new cases is arranged every year and 2,009 million people's death are arranged.In addition, there are millions of leprosy cases to cause by intracellular bacterial infection.Other that propagated by virulence factor in the cell cause weak property disease and comprise skin and visceral leishmaniasis, Chagas' disease (Chagas' disease), the Te Shi of Lee department disease, toxoplasmosis, histoplasmosis, trachoma, psittacosis, Q heat and the l that comprises the LegionnairesShi disease.At present, for the sensitive individual of these organisms of contact,, prevention dyes the measure that to take relatively seldom for causing weak sexuality.

Because the protection crowd human intrinsic M ﹠ M that avoids infected by tuberculosis and caused by tuberculosis effectively is one of most important disease that so tuberculosis is the mankind to be faced.More particularly, the people's pulmonary tuberculosis that is mainly caused by mycobacterium tuberculosis is a kind of major cause of developing country's patient death.Owing to can in scavenger cell and monocyte, survive,, infects mycobacterium tuberculosis so can producing in the chronic cell.By with itself hidden in mainly being responsible for detecting the cell of ektogenic and then activating immune system, mycobacterium tuberculosis can successfully be invaded the normal defense mechanism of host organisms relatively.Therefore these same cause of disease features have hindered the effective immunotherapeutic agent of resisting tuberculosis infection or the development of vaccine.Tubercule bacillus is cultivated relatively easily and observes under laboratory condition simultaneously.Therefore, mycobacterium tuberculosis is particularly suitable for proving principle and advantage of the present invention.

It will be understood to those of skill in the art that the mycobacterium tuberculosis of hereinafter discussing for example never is to be used for limiting the scope that the present invention handles mycobacterium tuberculosis.Equally, technology described herein is not to be limited to the treatment tuberculosis infection.On the contrary, can use the present invention to advantageously provide to be used to the immunologic determinants of the pathogenic factor that resists the outer product of any express cell and thereby suppress the safety that those organism infectivity propagate and the vaccine and the immunotherapeutic agent of product are arranged.

It is reported that the half of present nearly whole world population infects mycobacterium tuberculosis, cause annual millions of pulmonary tuberculosis cases that occur.Though this disease mainly be in Latin America, the developing country in Africa and Asia propagates, and in country of the first world bigger fashion trend arranged also., immunocompromised individuals particularly poor and also be in the precarious position of high-risk morbidity from the immigrant of high epidemic regions at the specific crowd of the U.S..Popular mainly due to acquired immune deficiency syndrome (AIDS) in developed country, present incidence lungy is also in continuous increase, and usually is to propagate with the form of multi-drug resistance mycobacterium tuberculosis.

Recently, the tuberculosis that resists one or more medicines has been reported in 36 states in 50 states of the U.S..In the New York, have 1st/3rd in all cases of check in 1991, anti-one or more main medicines.Though non-resistance tuberculosis can be cured through the life-time service microbiotic, the treatment prospect that relates to the drug resistance bacterial strain is still dim.Be subjected to the patient death rate of the strain infection of anti-two or more main microbiotic to be about 50%.Therefore, the safe and effective vaccine that needs against mycobacterium tuberculosis these mutation especially.

Because pathogenic agent still can continue survive several weeks or several months in wet or exsiccant phlegm, so the initial infection of mycobacterium tuberculosis almost always takes place by the particle that sucks aerosolization.Though former infection position is in lung, this organism also can infect bone, spleen, meninx and skin.According to the virulence of specific bacterial strain and the difference of host resistance, infect and can be very little or scope is very big the corresponding damage of tissue.In human case, the primary infection that great majority touch the individuality of toxic strain all is controlled.Reduced the propagation of bacterium in initial development of attacking the back acquired immunity, thereby made wound healing, most of people do not have symptom but have infectivity.

When mycobacterium tuberculosis not by when infected patient controls, usually cause the extensive degeneration of lung tissue.In sensitive individual, the damage in lung forms because tubercule bacillus breeds in alveolar or pulmonary macrophage usually.Along with the breeding of this organism, they can be diffused at a distance lymphoglandula and be diffused into the lung top by blood system by lymphsystem, marrow, kidney and hold the meninx of brain.Mainly due to cell-mediated hypersensitivity reaction, produce damage of characteristic granuloma or tuberculosis pro rata with the severity that infects.These damages are made up of monocyte, lymphocyte and fibroblastic epithelioid cell periphery.In most of examples, damage or tuberculosis finally necrose and stand dried network necrosis.

Though mycobacterium tuberculosis is a kind of important pathogenic agent, other bacterial classifications of Mycobacterium can cause that also animal comprises people's disease, and obviously are also included within the scope of the present invention.For example, the tuberculosis infection of domestic animals such as Mycobacterium bovis (M.bovis) and the closely related and responsible ox of mycobacterium tuberculosis, pig, sheep, horse, dog and cat.In addition, Mycobacterium bovis can pass through enteron aisle, the particularly infected person by pegnin.Respiratory tract is finally sent out in localized intestinal tract infections, occurs classical symptom lungy in short-term afterwards.The important pathogenic carrier of another of Mycobacterium is the Mycobacterium leprae of causing millions of ancient disease leprosy cases.Other mycobacterium species that cause the Animal diseases that comprise the people comprise mycobacterium kansasii (M.Kansasii), bird mycobacterium intracellulare (M.avium intracellulare), mycobacterium fortutitum (M.fortuitum), Mycobacterium marinum (M.marinum), Mycobacterium chelonei (M.chelonei), mycobacterium africanum (M.africanum), mycobacterium buruli (M ulcer-ans), mycobacterium microti (M.microti) and Mycobacterium scrofulaceum (M.scrofulaceum).The cause of disease mycobacteria strain its separately on DNA and the respective egg white matter sequence performance high homology is arranged, and some bacterial classification such as mycobacterium tuberculosis and Mycobacterium bovis are again closely-related.

Based on the moral reason of tangible practice, determine that in human body the experimental group compound is impracticable to the virgin work of the effectiveness of these slight illness.Therefore, because safety and expense, the ordinary method of developing in early days in any medicine or the vaccine is to use appropriate animal model.Based on recognizing that the immunodominance epi-position all is activated usually in host not of the same race, and infer the success of practical experimental animal model.Therefore, kind for example the immunogenicity determinant in rodent or the cavy generally not of the same race in as human body also be have immunoreactive.It is on probation only just to carry out the human clinical after having set up appropriate animal model fully, with further proof vaccine in intravital security of people and effectiveness.

With regard to the alveolar and the pulmonary infection of mycobacterium tuberculosis, guinea pig model very approaches the pathology of human body diseases in many aspects.Therefore, those skill in the art will appreciate that, the guinea pig model of this disease is prolonged shift the people onto and other Mammalss are suitable.The same with the people, it is responsive that cavy is dyed the tuberculosis sexuality of low dosage aerosolization human pathogen mycobacterium tuberculosis.Usually the people who is controlled with primary infection is different, and cavy is the sustainable development transmissible disease after contact aerosolization pathogenic agent, and help continue after analysis.In addition, cavy and presenting per capita to develop the tardy property skin hypersensitivity reaction that sclerosis of fine and close monocyte or the hardening region at the tuerculoderma position are feature.At last, the characteristic tuberculosis of people and cavy damage shows and comprises the similar morphology that has bright Han Shi giant cells.Because cavy is more responsive to primary infection than the people; and progression of disease is faster; so in the experiment of using this animal model, any provide protection of giving all will provide stronger indication for issuable same protective immunity in people or other not too responsive animals.Therefore, just to explaining rather than the restriction purpose that the present invention mainly will be proved as mammalian hosts with cavy.Those skill in the art will appreciate that other Mammalss of available people of comprising and domestic animal are realized the present invention.

Be subjected to the cause of disease carrier particularly in the cell any animal or human of infected by microbes all be difficult to excite host immune system.Though humoral response and corresponding protection production of antibodies can be controlled many infectants effectively, these mechanism mainly are that those are positioned in the health pathogenic agent in the extracellular fluid is effective.Particularly, opsonizing antibodies combines with extracellular adventitious agents, thereby makes them also kill and wound it then in cell to phagolysis is responsive.But to some other pathogenic agent then is not this situation.As if for example, former studies show that, humoral immune reaction does not play significant protective effect to the infection of bacterium in the anti-cell such as mycobacterium tuberculosis.Yet the present invention can produce the favourable humoral response to the target pathogenic agent, and like this, its effect promptly is not only limited to immunoreactive any special component of irriate.

As if more particularly, antibody-mediated defense reaction does not stop the primary infection of intracellular pathogen, in case and bacterium be sequestered in the host cell then will do not have defense function.As water soluble protein, antibody can pass extracellular fluid and blood, but is difficult to the lipid film migration by cell.In addition, in fact the generation of the opsonizing antibodies of antibacterium surface tissue may help intracellular pathogen to enter host cell.Therefore, effective preventive means of any anti-cell intrinsic factor such as mycobacterium tuberculosis all should add the cell-mediated immune response composition of a kind of aggressiveness, causing the lymphocytic rapid propagation of antigen-specific, the scavenger cell that is decreased so as to activation or remove it by cytotoxicity.But as what hereinafter will go through, the immune response of inducing cell mediation also is not equal to and induces protective immunity.Though cell-mediated immunity may be the prerequisite of protective immunity, produce vaccine according to technology of the present invention, also need to carry out the animal Attack Research.

This cell-mediated immune response generally comprises two steps.The first step is to finish the signal mark to infected cell with the specific molecular (main histocompatibility molecule or title MHC molecule) that the fragment of pathogenic agent can be transported on the cell surface.These MHC molecules combine with the small segment of the bacterioprotein of having degraded in infected cell, and it is presented in cell surface.Their immunity systems that presents stimulation of host on the T cell are so as to removing infected host cell or inducing host cell to eliminate and settle down on intracellular all bacteriums.

Different with most of infective bacterials, comprise that the mycobacterium species of mycobacterium tuberculosis tends in the vacuole internal breeding, and these vacuoles come down to the rest part sealed-off by film and cell.These protectiveness vacuoles of the natural formation of phagocytic cell and make them responsive especially to the infection of this class pathogenic agent.In such cavity, bacterium can avoid being degraded effectively, makes immunity system be difficult to present the bacterium composition of integration on the surface of infected cell.Yet the MHC molecule of infected cell will be shifted to vacuole and collect all free (release) bacterial product, perhaps shift to other positions of having carried external extracellular bacterial product in the host cell, so that normally present these products on cell surface.As former proof, the alien bacteria product present the appropriate reaction that will bring out host immune system.

Intracellular pathogen proposes relates to immune problem and has also constituted special challenge to vaccine development.For just, the anti-mycobacterial infections particularly production of the effective vaccine of Killing Mycobacterium Tuberculosis infection has made most researchers feel confused so far.At present, the extensive attenuated vaccine BCG (being strong virus force Mycobacterium bovis bacterial strain vaccine) that the vaccine of pathogenic agent is just lived in the anti-cell that utilizes, with it as proactive tool to anti-tubercle bacillus.In 1988, the further investigation that the World Health Organization carries out in India determined, the efficient of best BCG vaccine also so low so that detect less than.Although debatable like this effectiveness is arranged, the BCG vaccine still is extensive use of in whole world tuberculosis hotspot always.More serious problem is, even the people who has inoculated BCG also usually produces the susceptibility to tuberculin, thereby negates the availability that is used for carrying out the most frequently used tuerculoderma of tuberculosis screening and control.

Relating to use attenuated vaccine alive such as another serious problems of BCG is possible cause the mortality disease in the immunocompromised patient body.These vaccines can cause a kind of special danger to the people that the cell-mediated property immunity of being constrained is arranged, and this is because they have reduced the ability of the infection that resists rapid proliferation-inducing.Such individuality comprises because of the malnutritive and abominable weakly people of living condition, organ transplantation recipient, and is subjected to HIV the infected.Just use the BCG vaccine, the high-risk individuality also comprises suffering from lung's illness such as pulmonary emphysema, chronic bronchitis, pneumoconiosis, silicosis or the tuberculosis patient was arranged in the past.Therefore, should be only limited to the vaccine that in having the special population of maximum potential income, uses attenuation.

Use the attenuated vaccine that lives also may produce the side effect that other are not expected.Because living vaccine can be at recipient's internal regeneration, so they can produce the more antibody of wide region and littler cell-mediated specific aim immune response than non-infectious vaccine.This shotgun usually can be blocked at those the most normal immune responses of participating in the molecular structure of cell prophylactic effect.In addition, using has the living vaccine of complete film structure can induce the generation opsonizing antibodies, so as to making the required exosome of a kind of effective prevention.Therefore, after the host touched the toxicity strain of target microorganism, in fact the existence of these antibody can increase the absorption of non-attenuated pathogens in host cell, and survived therein and breed.Moreover the vaccine of attenuation contains thousands of kinds of different molecules and thereby more may contain deleterious or can cause unfavorable immunoreactive molecule in the patient body.The other problems relevant with using living vaccine comprises Poison Reverse, self-sow contact, Virus Pollution and virus interference, and is difficult to realize stdn.

Equally, non-infectious vaccine is restricted aspect the bacterium in suppressing cell as dead microorganism or conventional s-generation subunit vaccine at strong antigen film integrated structure.The same with attenuated vaccine, the bacterium that kills also causes the generally reaction that can suppress most of effective preventative determinants.In addition, killed vaccine still exists in a large number at immune potential antigenic structure, thereby increases the possibility of toxic reaction or regulation of immune system effect.The traditional subunit vaccine (no matter being synthetic or purifying) that comprises the film integrated structure also may be induced strong opsonization, enters scavenger cell and breeding therein and help intracellular pathogen.The antigenic killed vaccine of anti-cell internal surface can increase the relative virus force of pathogenic factor by the speed that the increase bacterium is forgiven.Therefore, under the situation of intracellular pathogen, may be unsuitable for using the conventional attenuation or the killed vaccine of anti-strong antigen bacterium surface composition.

For fear of with use the relevant problem of traditional vaccine, used extracellular protein or its immunogenicity analogue to develop new vaccine, so as to the protective immunity of stimulation at pathogenic agent in the specific cell.For example; the inventor's U.S. Patent No. 5; 108,745 (announcements on April 28th, 1992) disclose the vaccine and the method for the protective immunity that produces anti-close lung legionella (Legionella pneu-mophila) and mycobacterium tuberculosis and other intracellular pathogens.These existing vaccines always are based on the extracellular products that is derived from protein compound at first, said protein compound is discharged in the vitro culture to the extracellular by pathogenic bacteria, and discharge to the extracellular by the bacterium in the infected host cell in the body.As disclosed in the literary composition, these vaccines are based on selectivity and identify that those can stimulate strong immunoreactive extracellular products or its analogue of target pathogenic agent in the resisting mammal host to make.

More particularly, by detecting it causes strong lymphproliferation response or skin delayed type hypersensitivity in the mammalian body that pathogenic agent interested is had immunity ability, screen the outer protein of these existing candidate cells.Though this disclosed method and relevant vaccine have been avoided many use traditional vaccine inherent shortcomings, complicated the supporting of the selection of effective immunological reagent cut the immune response effect because cross reactivity and host's change may make.Therefore, though molecular immune originality is one of index of effective vaccine, some other factor then may make it cause in vivo and produce insoluble problem in the effective immune response.

The more important thing is, be surprisingly found out that,, identify effective protective immunity to induce the conventional art methods of vaccine be very inconvenient and may be invalid particularly with regard to mycobacterium tuberculosis.For example, mycobacterium tuberculosis extracellular protein in enormous quantities is carried out SDS-PAGE analyze, the purpose of carrying out conventional Western engram analysis then is to identify in these extracellular compositions the immunogenic part of tool, and the result has produced inconsistent result.The test of having reported does not have to identify comes out should produce the extracellular products of strong immunogenic response, and thereby consistent with original idea, just do not bring into play its function as effective vaccine yet.Many extracellular products of mycobacterium tuberculosis all are known in the art, have done to identify and some has also measured sequence.In addition, the same with any extraneous protein, can show that these known compounds have induced immune response.Yet this area does not have evidence to show that directly these known compounds can induce the protective immunity of identifying as tradition.

Therefore, a main purpose of the present invention provides vaccine or immunotherapeutic agent and production method and the application in the effective immune response that causes the infectivity resistant bacterial pathogens, and it does not also rely on that traditional vaccine is considered and based on the selection technology of high specific, strong immunogen operability.

Another object of the present invention provides vaccine or immunotherapeutic agent and be used for giving the method that antagonism comprises the intracellular pathogen acquired immunity of mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium kansasii, bird mycobacterium intracellulare, mycobacterium fortutitum, Mycobacterium chelonei, Mycobacterium marinum, mycobacterium africanum, mycobacterium buruli, mycobacterium microti and Mycobacterium scrofulaceum in the mammalian hosts body.

A further object of the present invention provides vaccine and the immunotherapeutic agent that is easy to generate.It has the toxicity of reduction with respect to killed vaccine or attenuated vaccine performance.

The summary of invention

The present invention has realized above-mentioned and other purpose by compound and production method thereof as vaccine and/or immunotherapeutic agent are provided with the protectiveness or the therapeutic immune response that produce the antipathogen infection in the mammalian hosts body.Generally speaking, the invention provides the protectiveness of inducing antagonism extracellular compound generative nature infection carrier or the means of therapeutic immune response.Though compound antagonism pathogenic bacteria of the present invention is effective especially, they can be used for producing protectiveness or therapeutic immune response, generate any pathogenic agent of mainly enriching extracellular products with antagonism.

For purposes of the invention, term " main abundant " is interpreted as one and assert that those are by the relative terms of interested pathogenic agent with the extracellular products of maximum release.For example, be about with regard to 0.5 the mycobacterium tuberculosis with regard to grow to optical density(OD) under various different culture condition, those skilled in the art are expected to obtain the 10 μ g/mL orders of magnitude or more mainly enrich extracellular products.Therefore, in the ultimate production of the extracellular products of 4mg/L altogether of the mycobacterium tuberculosis of growing under normal or heat-shocked condition, about 15 to 20 parts (independent or merging) in about 100 parts of known extracellular products will constitute about 90% of total amount.These are that expectation is included in and mainly enriches extracellular products in the scope of the invention, and are easy to be discerned as the broadband that occurs in the SDS-PAGE gel.In addition, can further identify interested extracellular products and distinguish it according to the amino acid sequence analysis result.Remaining extracellular products is fewer.Those skilled in the art it will also be appreciated that the difference according to culture condition, and the relative quantification abundance of the main extracellular products of specificity can change to some extent.Yet in most of the cases, the identification of mainly enriching extracellular products individually will can not change.

Therefore, the present invention can be used for protecting mammalian hosts not to be subjected to virus, bacterium, fungi or protista thing pathogenic infection.Should be mentioned that in some cases as in virus infection, can produce by infected host cell and mainly enrich extracellular products.All discharge the microorganism mainly enrich extracellular products though antagonism is arranged, and vaccine of the present invention and method pathogenic agent in producing anti-cell comprises that in the protective immunity of not of the same race and serotype mycobacterium tuberculosis be effective especially.Vaccine of the present invention also is effective as the immunotherapeutic agent of the existing disease of treatment.

The inventor is surprisingly found out that, uses by discharge the abundantest of bacterial pathogens extracellular or mainly enriches product or its immunogenicity analogue carries out immunity and can cause effective immune response, and do not consider the absolute immunity originality to drug compound.Since they be in microbe, discharge and thereby can be used to participate in antigen processing by host's molecule and present, and because they are in the natural high density in tissue between period of infection, so the extracellular products that mainly enriches of pathogenic factor is subject to processing and is presented to host immune system than other bacterium compositions are more normal.With regard to intracellular pathogen, mainly enrich extracellular products be presented on the infected host cell surface the main immunogenic determinant and therefore and around in the environment performance bigger amount is arranged.Therefore, the acquired immunity permission host defense system that mainly enriches extracellular products of resisting pathogenic microbes detects rapidly and is isolated in the pathogenic agent in the host cell and suppresses them effectively.

More particularly, by pathogenic bacteria discharge mainly enrich product seemingly by phagocytic cell and other host immune system mechanism with than speed processing not too general or that membrane-bound cause of disease composition is bigger, and no matter they separately immunogen activity or specificity how.During bacterium, this immunity processing discordance is a particularly important in pathogenic factor is the isolated cell of a kind of and normal immunocompetence.Be presented to infected host immune system in a large number and continuously by means of them, the outer product of bacterial cell or its immunogenicity analogue can cause the intensive immune response the most widely, and totally unnecessaryly take their molecular immune originality features separately into account.

Mainly enrich extracellular products and be by the target pathogenic agent and be discharged into the protein in the surrounding environment and the main composition composition of other molecular entities.Nearest studies show that, single in some cases mainly enrich extracellular products can account for microorganism release product reach 40% (weight).More commonly, mainly enrich individually extracellular products and amount to about 0.5% to 25% of the gross product that accounts for infectious agent release.In addition, can find maximum 5 kinds or 6 kinds mainly enrich extracellular products account for microorganism release total mass 60% to 70%.Certainly, those skill in the art will appreciate that with regard to releasable absolute or relative product amount, the level relatively of extracellular products may fluctuate to some extent in different time.For example, pH, oxygenant, perviousness, heat and all can change the composition and the amount of the product that is discharged to the moiety of different steps, reproduced state and the surrounding environment of other stress conditions of organism, biological cycle.Moreover between different strain even between the different strains in certain kind, the absolute and relative level of extracellular products has a great difference.

With regard to intracellular pathogen, extracellular products seems the extendible specific immunity lymphocyte population that can detect and bring into play the anti-microbial effect of the anti-scavenger cell that contains bacterium alive.In addition, be presented in the lip-deep ability of infected cell repeatedly by them, main abundant or main extracellular products can have the function of effective antigen mark.Therefore; according to technology of the present invention; carry out vaccine inoculation and induce at pathogenic bacteria mainly enrich the protective immunity that extracellular products or its immunogen are equal to determinant, when continue after promote host immune system to set up rapidly and effective immune response when being subjected to the target pathogenic infection with strong cell-mediated composition.

Produce vaccine and come the prior art immunity of immune response stimulating movable opposite with mainly concentrating on based on the molecular immune originality of the high degree of specificity of the pathogenic agent composition of indivedual screenings; the outer product of bacterial cell or its immunogenicity analogue (rather than their immunogen specificity) that preferential development utilization of the present invention is abundant relatively are with in fact having the specific compound of lower immunogen to set up or induce protective immunity than not too general extracellular products performance.With regard to the disclosure; the immunogen analogue is to be enough to mainly enrich extracellular products or its part similar in appearance at least a by what the target pathogenic agent was expressed, have continue after stimulate any molecule or the compound of the protective immunological reaction in the mammalian hosts body when being subjected to the target pathogenic infection.Briefly, vaccine of the present invention is by selecting mainly to enrich product (maybe can stimulate substance to be equal to immunoreactive molecule analogue) and identifying or producing with pure relatively isolated in form by special pathogen to what the extracellular discharged.Can use technology known in the art to prepare one or more isolating immunoreactivity products then, and being subjected to target immune mammalian hosts before pathogenic infection, to cause the preventative immune response of required anti-target pathogenic agent.

What can predict is, vaccine of the present invention will by at least one, two or may in addition several fully immunogenic determinant of qualification form.Thereby the present invention can produce constant, standardized vaccine, and it can be developed, test and relatively easily and promptly use.In addition, use the molecule of the several abundant qualifications be equivalent to mainly to enrich secretory product or extracellular products can reduce the harmful side effect relevant greatly, and can eliminate possible occlusion effective immunogen sign with conventional vaccine.Equally because vaccine of the present invention is not a vaccine a kind of attenuation or that kill, so produce, during the purifying or after administration, can eliminate the danger of infection effectively.Therefore, vaccine of the present invention can be used for the individuality of immunocompromised host safely, comprises asymptomatic tuberculosis patient and is subjected to the patient that HIV infects.Moreover, because humoral immune reaction is unlimitedly at by the product of target pathogenic agent release, so almost there is not to produce the chance of harmful opsonic immunity composition.Therefore, the present invention can make the humoral response of irriate help from antibody sensitizing range removing target pathogenic agent.

Another favourable aspect of the present invention be can so as to gather in the crops with comparalive ease, produce and continue after purified vaccine.For example, the available less workload abundant extracellular products of from the target pathogen culture thing that comprises mycobacterium tuberculosis or Mycobacterium bovis, gaining the upper hand.Because required compound is released in the substratum, separate it at an easy rate being divided into so can utilize routine techniques in bacterial body, to be combined into the film of target pathogenic agent during microorganism growth.Be more preferably and from genetic engineering modified organism, produce the also required immunoreactivity composition of purifying vaccine of the present invention, cloned the gene of expressing the certain detail extracellular products of mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae or other pathogenic agent interested in the wherein said organism.As known in the art, can modify these through engineering approaches organisms and make it to produce the extracellular products of higher levels of selection or modified immunogenicity analogue.Perhaps, can use technology known in the art chemically synthetic immunoprotection product, its part or its analogue.No matter the production that utilized source how, all can use fractional separation, chromatography or other purification process to learn and biochemical means commonly used such as conventional compounding process are separated advantage or mainly enriched the immunogenic components of extracellular products and be mixed with releasable vaccine thereafter.

For example, in an illustrative embodiment of the present invention, the target pathogenic agent is a mycobacterium tuberculosis, isolates to be discharged into by mycobacterium tuberculosis from other bacterium compositions and mainly enriches the extracellular in the nutrient solution, and product also is used to cause the immune response of mammalian hosts.In based on the attack of animal experiment, utilize indivedual protein or class protein to identify that those can induce protective immunity then, and make it to be suitable for according to the protein of technology of the present invention as vaccine.More particularly, after cultivating and gathering in the crops bacterium,, in bacterium He other compositions, isolate main extracellular products by centrifugal and filtration by its physics abundance.In case of necessity, re-use ammonium sulfate precipitation method resulting a large amount of filtrates are carried out fractional separation, that continues dialyses with the mixture of the extracellular products that obtains being commonly referred to as EP.Use suitable chromatographic technique known in the art and hereinafter more abundant description to be purified to substantial homogeneous state then through the dissolved extracellular products in the part of dialysis.

These methods of giving an example are produced 14 kinds of one molecular weight at the main extracellular protein product of the mycobacterium tuberculosis of 110 to 12 kilodaltons (KD).Each single extracellular products that mainly enriches shows one and is equivalent to its band of molecular weight separately behind the purifying on polyacrylamide gel electrophoresis, thereby identified and prepared to be equivalent to mainly enrich indivedual products of extracellular products or respectively to organize product, to be used as vaccine of the present invention.Can use their all or part of aminoacid sequences separately of the general technology for detection in this area with further qualitative and distinguish purifying mainly enrich extracellular products.Sequential analysis also can be the possible structural relation that understanding mainly enriches between extracellular products information is provided.

Then, can be in for some time through the extracellular products of subcutaneous or these purifying of intradermal injection repeatedly, by technology immunity Mammals of the present invention and at mammalian hosts system moderate stimulation acquired immunity.For example; that injects that one or more are added on purifying in the incomplete Freund's adjuvant mainly enriches the outer product of bacterial cell; be added in then and carry out second time injection in the same adjuvant, about three Zhou Houke are in order to cause at the protective immunological reaction after the back is with the attack of toxicity pathogenic agent.Other exemplary immunization methods in the scope of the invention and the technology can comprise that injection within a certain period of time is added in extracellular products or its analogue of the purifying among the Syntex Adjuvant Formulation (SAF), injects 3 or 4 times altogether.Though series injection is generally more effective, what once come into operation and select mainly enriches extracellular products or its immunogenicity subunit or analogue and also can produce the immune response of expection, and expects that this method is also included within the scope of the present invention.

Can use the experimental model such as the cavy of approval to prove the immunization method that these are given an example.Example as will be discussed in more detail below, be added in the bacterial product in the SAF adjuvant and set up the corresponding false immunity of control animal through three injections, share five kinds of immunity of mainly enriching extracellular products (press preceding method from mycobacterium tuberculosis purifying) to several cavys to finish.Each proteinic dosage range is 100 μ g to 2 μ g.After the last immunization, make all animals aerosolization mycobacterium tuberculosis of contagiousnesses and potential lethal dose and long-time monitoring simultaneously.With share the tuberculosis branch mutually the animal of mainly enriching the extracellular products immunity of bacterium compare, control animals shows tangible weight loss.In addition, at viewing duration the death of half control animals is arranged, and all animals through immunity all do not die from tuberculosis.The necrotomy of carrying out after the experiment shows that non-immune control animals has corresponding damage in more significant colony forming unit (CFU) and its lung and the spleen than protected species.Test predicts the extracellular products that mainly enriches that share other 17 kinds of purifying and has immunoprophylactic effect, thereby proves scope of the present invention, and can prepare the vaccine of wide antibacterial range according to technology of the present invention.

But what should emphasize is that the present invention is not limited to share secretory product or extracellular products.For example, several experimental techniques that substitute prove that according to technology of the present invention, the single extracellular products that enriches promptly has the ability of inducing Mammals to produce protective immunity.In each experiment, the chromatography method purifying from mycobacterium tuberculosis that uses this paper to describe in detail is single mainly to enrich extracellular products and in order to immune guinea pig.An example is to use the adjunvant composition that enriches secretory product (molecular weight is equivalent to 30KD) that contains purifying to inoculate animal in multiple experiment.In another embodiment of the present invention, use contains the isolating different cavy of adjunvant composition immunization of enriching extracellular products (molecular weight is equivalent to 71KD) from mycobacterium tuberculosis.After the immunity, make two groups of laboratory animal contact the aerosolization mycobacterium tuberculosis of lethal dose with suitable control animals to determine vaccine potency.

More particularly, in once testing, 6 cavys of three usefulness 100 μ g 30KD protein (being added among the SAF) immunity in 6 time-of-weeks.Use the batch formulation or the damping fluid inoculation control animals of the extracellular protein (EP) of respective amount simultaneously.3 weeks of last inoculation back are with the aerosolization mycobacterium tuberculosis attack animal of lethal dose and monitored for 14 weeks.The cavy survival rate of the outer product system system immunity of the cavy of 30KD protein immunity and whole cell is respectively 67% and 50% and (illustrates with respect to EP, mainly enrich the unpredictable good usefulness of extracellular products), the animal of false immunity then has only 17% survival rate.After experiment stops, the tubercule bacillus that kill animals and check are lived.As seen the result is presented at the mycobacterium tuberculosis that remarkable high density is arranged in lung and the spleen without the animal of immunity.

Those animals with the immunity of 71KD protein are carried out similar experiment.In the experiment, in three time-of-weeks, use 6 cavys of twice immunization of the proteinic SAF adjunvant composition of 71KD that contain 100 μ g purifying.Other animals then do similar immunity with the whole preparation of the extracellular protein of purifying not or as the EP of positive control and as the damping fluid of negative control.Behind the contact lethal quantity aerosolization tubercule bacillus, monitoring cavy body weight 6 months.Produced the protective immunity of anti-strong virus force mycobacterium tuberculosis once more with the animal of enriching the extracellular products immunity of purified form.This stage at the end compares with the experimental group animal, shows tangible body weight with the animal of damping fluid immunity and reduces.In addition, the animal dis motility rate of positive controls and the immunity of 71KD protein is respectively 63% and 50%, and all dead before the observation period finishes without the animal of immunity.

Preparation that it should be noted that vaccine is not critical for the present invention, and can make optimizing and change and to be beneficial to administration.Can use separately or unite use in any mode that designs and mainly enrich the solution of the immunogenicity determinant of the purifying that extracellular products makes, to produce protective immunological reaction from pathogenic agent.Can come into operation the separately protein soln of purifying is perhaps with administration after itself and the adjuvant mixed preparing.Being used for the present invention is SAF, the adjuvant that contains monophosphoryl lipid A, Freund's incomplete adjuvant and the Freund's complete adjuvant that contains dead bacterium with the active particular exemplary adjuvant that improves the immunogenicity determinant of selecting.Can be used for other adjuvants of the present invention is water-in-oil emulsion, mineral salt (for example alum), nucleic acid, block polymer tensio-active agent and microorganism wall (peptidoglycolipid).Though do not limit the scope of the invention, believe that antigen discharges from the injection site because adjuvant can slow down, so may improve immune response.

Those skilled in the art are at the accompanying drawing in conjunction with brief description that will at first provide hereinafter, read following detailed description of preferred embodiments of the invention after, will fully understand other purposes of the present invention, feature and advantage.

Brief description of drawings

Fig. 1 shows 4 gels that are denoted as the Coomassie blue stain of 1A to 1D, illustrates as mainly enrich through several mycobacterium tuberculosis that SDS-PAGE (SDS-PAGE) is identified the purifying of extracellular products.

Fig. 2 shows that 12 kinds of mycobacterium tuberculosis of evaluation mainly enrich 5 N-terminal amino acid of extracellular products, and the apparent molecular weight of 14 kinds of these class products.

Fig. 3 tabulates and shows that three kinds of mycobacterium tuberculosis mainly enrich the rapid aminoacid sequence in N end of the extension of secretory product, and these products are to distinguish according to 5 rapid amino acid in N end shown in Fig. 2.

Fig. 4 is that diagram is relatively mainly enriched the cavy of secretory product immunity, the positive control treated animal of the outer protein formulation immunity of usefulness prior art whole cell with the mycobacterium tuberculosis 30KD of purifying, and the survival rate of non-immune negative control treated animal behind contact lethal quantity aerosolization mycobacterium tuberculosis.

Fig. 5 is the extracellular products immunity is relatively mainly enriched in diagram with the 71KD of purifying a cavy, with the positive control treated animal of the outer protein formulation immunity of mycobacterium tuberculosis whole cell in the prior art, reach the average cavy body weight of negative control treated animal behind contact lethal quantity aerosolization mycobacterium tuberculosis without immunity.

Fig. 6 be in the diagram comparison diagram 5 with the 71KD of the purifying of mycobacterium tuberculosis mainly enrich the cavy of extracellular products immunity, with the positive control treated animal of the whole preparation immunity of extracellular protein in the prior art, and the survival rate of non-immune negative control treated animal behind contact lethal quantity aerosolization mycobacterium tuberculosis.

Fig. 7 is the diagram comparison in second time different experiments with the cavy of mainly enriching the immunity of 71KD extracellular products of purifying and mean body weight after non-immune negative control treated animal is contacting lethal dose aerosolization mycobacterium tuberculosis.

Fig. 8 A and 8B are that diagram is relatively at PPD ⁺(indication of m tuberculosis infection) and PPD ^-In the human body to the lymphproliferation response that mainly enriches the 71KD extracellular products of the purifying of giving an example.Fig. 8 A is shown in lymphocyte and this antigen is incubated the back numerical value that recorded in 2 days, and Fig. 8 B is shown in the back numerical value that recorded in 4 days of insulation.

Fig. 9 is that diagram is relatively with the cavy and the mean body weight of non-immune control animals after contacting lethal quantity aerosolization mycobacterium tuberculosis that contain by the vaccine immunity of the extracellular products of the associating of the technology of the present invention production.

Figure 10 is that diagram is relatively with the cavy by the vaccine immunity of the extracellular products of the associating of the technology of the present invention production of containing of three various dose, with the mean body weight of non-immune control group cavy after contacting lethal quantity aerosolization mycobacterium tuberculosis.

Figure 11 is that diagram is relatively with the cavy by the vaccine immunity of the extracellular products of the technology of the present invention production that contains with six kinds of different modes combinations, with the mean body weight of control group cavy after contacting lethal quantity aerosolization mycobacterium tuberculosis of non-immunity.

The detailed description of invention

The present invention relates to compound and its production method and as the application of vaccine and immunotherapeutic agent antagonism Pathogenic organisms.More particularly; the present invention relates to the production of mainly enriching extracellular products or its immunogenicity analogue that discharges by Pathogenic organisms and as the application of vaccine or immunotherapeutic agent, and the methods involving that in mammalian hosts, produces anti-infective protective immunity.For ease of describing, among the application these compounds are called vaccine.

In the embodiment of the present invention of illustrative, distinguish and subsequent purificn mycobacterium tuberculosis mainly enrich extracellular products.With these main advantage extracellular products immune guinea pigs of purified form, and needn't determine the special molecular immunogenicity of indivedual products.In addition, can individually or unite the extracellular products that uses purifying, and carry out immunity with different dosage and route of administration.Those skilled in the art will be understood that, can utilize above-mentioned strategy to go to put into practice method of the present invention with any Pathogenic organisms or bacterium, so the present invention are not limited to against mycobacterium tuberculosis vaccine and method especially.

In the embodiment that these are given an example, use column chromatography to separate and the purifying mycobacterium tuberculosis mainly enrich extracellular products.Use polyacrylamide gel electrophoresis determine the relative abundance of extracellular products and purifying it.Behind the purified vaccine composition, separately or unite use and mainly enrich extracellular products immunization cavy, and attacking animal with mycobacterium tuberculosis thereafter.As will be discussed in more detail below, but except the detection reaction of the expection of after immunity, setting up anti-these extracellular products, the also unpredictable ground of vaccine of the present invention in these laboratory animal bodies, cause antagonism continue after effective immunity of lethal quantity aerosolization m tuberculosis infection.

Though these embodiments of giving an example have been used the extracellular products of purified form, it will be understood by those skilled in the art that to use and realize the present invention at an easy rate by the immunogen analogue of recombinant technology production or with the other forms of extracellular products of technology chemistry synthetic known in the art.In addition, can use the fragment of immunogenicity analogue, homologue or its selection of mainly enriching extracellular products to replace belonging to the scope of the invention and the interior natural product of principle.

For making those skilled in the art further understand the present invention, provide following non-limiting examples, to illustrate evaluation, separation, produce and to use the method for mainly enriching extracellular products (separately and unite use) as vaccine.

Embodiment 1

The outer protein (EP) of whole cell in separation and the production mycobacterium tuberculosis

(American Tissue Culture Collec-tion, Rockville Md) obtain mycobacterium tuberculosis Erdman bacterial strain (ATCC 35801) from U.S. tissue culture preservation center.With freeze dried bacterium be suspended in again Middlebrook 7H9 substratum (DifcoLaborato-ries, Detroit, Mich.) in and remain on the Middlebrook7H10 nutrient agar.Use Bacto Middlebrook 7h10 agar (Difco), OADCEnrichment Medium (Difco), 0.1% casein enzymatic hydrolyzate (Sigma) and glycerine to prepare 7H11 agar (Cohn by the preceding method of having described, M.L., Am.Rev.Respir.Dis., 98:295-296).Behind the autoclaving, agar is interspersed among Micro-Organism Culture Dish (in 100 * 15mm) and cool off it.

Use Aseptic technique shop to apply mycobacterium tuberculosis and then at 5%CO ₂-95% air, 37 ℃ of cultivations in 100% humidity environment.After cultivating 7 days on the 7H11, scrape from plate and to get bacterium colony, with 10 ⁸CFU/ml be suspended in the 7H9 nutrient solution and be sub-packed in 1.8ml Nunc freeze pipe (Roskilde, Denmark) in.With 998ml distilled water and 2ml glycerine (SigmaChemical, Co., St.Louis, MO.) rehydrated 7.7g Bacto Middlebrook 7H9 powder to be to prepare every liter of nutrient solution, then mixture is transferred to pH6.75 and with nutrient solution in 121 ℃ of autoclavings 15 minutes.The cell of slow freezing equal portions and be stored in-70 ℃ then.Still but indefinitely, survival also can be used it to the cell of Zhu Cuning when needed under these conditions.

Mycobacterium tuberculosis culture from grow in above-mentioned Middlebrook 7H9 nutrient solution makes protein outside the whole cell (EP) preparation.Again after the preparation, under 121 ℃,, and be scattered in the Co-star225cm of emptying with the nutrient solution autoclaving of 150ml equal portions 15 minutes ²In the tissue culture flasks.Thawing is by the mycobacterium tuberculosis cell under mentioned above being stored in-70 ℃ and be used to inoculate the 7H11 agar plate.Cultivate after 7 days, scrape from plate and get bacterium colony, be suspended in a few ml 7H9 nutrient solutions, and in water-soluble, carry out supersound process to form single cell suspension.According to Perkin-Elmer Junior 35 type spectrophotometers (Norwalk, Conn) detected result, initial light density be 0.05 o'clock with the aseptic suspension mycobacterium tuberculosis of 150ml equal portions cell.Then at 5%CO ₂In-95% air with cell in 37 ℃ of 3 weeks of insulation, show 0.4 to 0.5 optical density(OD) until suspension.With these cultures as the original bacteria liquid of further in the 7H9 nutrient solution, cultivating.The supersound process original bacteria liquid makes it to form single cell suspension in water-soluble.In the 7H9 nutrient solution, be 0.05 then with mycobacterium tuberculosis cell dilution to initial light density, and at 5%CO ₂Be incubated 2 in 37 ℃ in-95% air ¹/ ₂-3 weeks were 0.4-0.5 up to optical density(OD).Separation and Culture thing supernatant also passes through 0.8 μ m and 0.2 μ m lower protein in succession in conjunction with filter membrane (Gelman SciencesInc, Am.Arbor, Mich.) filtration sterilization.In the Filtron Minisette ultra-fine filter that has 10KD molecular weight cut-off Omega film, above-mentioned filtrate is concentrated about 35 times then, and 4 ℃ of down storages.(SDS-PAGE) analyzes the protein composition that protein formulation outside the whole cell discloses multi-ribbon through sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Obtain the culture filtrate of 40-95% ammonium sulfate precipitation, make the outer protein mixture (EP) of whole cell.

Embodiment 2

The purifying mycobacterium tuberculosis substantially mainly enrich extracellular products

(the I level Sigma), and stirs gently with fractional separation protein to add ammonium sulfate with 10% to 95% concentration in the sterile culture thing filtrate of embodiment 1 under 0 ℃.To suspend moves in the Plastic Bottle, with RC3B Sorvall whizzer in major diameter charging basket rotor 3, the centrifugal throw out of 000rpm with resulting separation.Pour out supernatant liquor and be further purified supernatant liquor or precipitated product according to required product difference.When interested product is contained in the supernatant liquor,, finish the ammonium sulfate precipitation second time to supernatant liquor through increasing the above-mentioned first time of sedimentary salt concn.Centrifugal as stated above precipitating required product after stirring solution gently, and second supernatant liquor is carried out further purifying.

After centrifugal, again be dissolved in sedimentary protein in the suitable cold buffer liquid and at (the Spectrum Medi-cal Industries of the Spectrapor dialysis membrane with 6,000 to 8,000 molecular weight cut-offs, Los Angeles, California) in thoroughly dialysis with desalination.(Pierec Chemical Co., Rockford Illinois) detect extracellular protein concentration, and use SDS-PAGE to detect the each several part composition with two cinchonic acid protein detection methods.Each several part is added on the chromatography column is further purified it.

Use above concise and to the point general flow, outside the general cell that makes by embodiment 1 described method, be purified into 14 kinds of extracellular products the protein filtrate.Below definite ammonium sulfate precipitation method and the chromatography method of the isolating every kind of extracellular products detailed description of institute just.A.110KD extracellular products

1. obtain 50-100% ammonium sulfate precipitation thing as stated above.

2. dialyse again the dissolved throw out and be splined on DEAE Sepharose CL-6B or QAE Sepharose ion exchange column on, its center pillar damping fluid is made up of 10% sorbyl alcohol, 10mM potassiumphosphate (pH7), 5mM 2 mercapto ethanol and 0.2mM EDTA, and uses the sodium-chlor gradient elution.Wash-out contains the proteinic part of 110KD and collects it when about 550mM salt concn.The S200 Sepharose size that the part of collecting is splined in PB5 (phosphate buffered saline (PBS)) is divided in the receipts separator column.As this protein of homogeneous 110KD protein wash-out.B.80KD extracellular products

1. discard 0-25% ammonium sulfate precipitation part (0 °, 1 hour) and keep 25-60% ammonium sulfate precipitation part (0 ℃ is spent the night) by method discussed above.

2. with 25mM Tris (pH8.7) the filling DEAE CL-6B post (pHarmacia) that contains 1M NaCl, and with 25mM Tris (Ph8.7), 10mM NaCl balance it, with protein example to 25mM Tris dialysis also application of sample on post.Wash post with same damping fluid and spend the night, make be added in 10mM to the 200mM NaCl among the 25mM Tris (pH8.7) first salt gradient by post with other protein of wash-out.Make second salt gradient (200 to 300mM NaCl) by post and at the about wash-out 80KD of 275mM NaCl place protein.

3. with 25mM Tris (pH8.7), 10mM NaCl dress Q-Sepharose HP post also equilibrates to 25mM Tris (pH8.7), 10mM NaCl again.Protein example is dialysed and upper prop to 25mM Tris (pH8.7), 10mM NaCl.In same damping fluid, wash post, then with the 200-300mM NaCl wash-out that is added among the 25mM Tris (pH8.7).

4. collect and contain the proteinic part of 80KD and, in the Speed-Vac thickener, be concentrated to 1-2ml then 25mM Tris (pH8.7), 10mM NaCl dialysis.The protein example application of sample is also washed post with 25mM Tris (pH8.7), 150mM NaCl on Superdex 75 posts.As homogeneous protein wash-out 80KD protein.C.71KD extracellular products

1. make 40-95% ammonium sulfate precipitation thing as stated above, but different be in 7H9 nutrient solution (pH7.4), in 0%CO ₂Environment is cultivated down the 71KD product, and in 42 ℃ of heat-shockeds 3 hours, weekly.With throw out to initial buffer liquid (InitialBuffer; 20mM Hepes, 2mM MgAc, 25mM KCl, 10mM (NH ₄) ₂SO ₄, 0.8mM DL-dithiothreitol (DTT), pH7.0) dialysis.

2. incite somebody to action again dissolved throw out application of sample on the ATP A-garose post that initial buffer liquid balance is crossed.Collect effluent and go up ATP Agarose post once more.The 71KD protein bound is on post.

3. at first use initial buffer liquid, use 1M KCl again, wash the ATPAgarose post with initial buffer liquid then.

4. with 10mM ATP wash-out homogeneous 71KD protein and phosphate buffered saline buffer dialysed from the post.D.58KD extracellular products

1. obtain 25-50% ammonium sulfate precipitation thing as stated above.

2. dialyse again the dissolved throw out and be added on DEAE-SepharoseCL-6B or the QAE-Sepharose post on, use the NaCl wash-out then.What be collected in about 400mM NaCl place wash-out contains the proteinic part of 58KD.

3. make the part of collection cross Sepharose CL-6B post then.At about 670-700,000 dalton locates this protein of wash-out.

4. with thiopropyl-agarose column on the protein of wash-out.At the about wash-out homogeneous 58KD of 250-350mM2-thioglycol place protein.Use the protein of SDS-PAGE monitoring wash-out and on gel, show list-band shown in Figure 1A the 2nd hurdle.E.45KD extracellular products

1.a. discard 0-25% ammonium sulfate precipitation thing (0 ℃ following 1 hour).

B. keep 25-60% ammonium sulfate precipitation thing (spending the night under 0 ℃).

2.a. also use 25mM Tris, this post of 10mM NaCl (pH8.7) balance with 2.5mM Tris (pH8.7) the dress DEAE CL-6B post (Pharmacia) that contains 1M NaCl.

B. with protein example to 25mM Tris, 10mM NaCl (pH8.7) dialysis and upper prop.Washing post with same damping fluid then spends the night.

C. with being added in 25mM Tris, salt gradient (10mM to 200mM) the wash-out post in the pH8.7 damping fluid.At the about wash-out 45KD of 40mM NaCl place protein.

3.a. with the 25mM Tris that contains 1M NaCl, pH8.7 fills Q-SepharoseHP post (Pharmacia) and uses 25mM Tris once more, 10mM NaCl, the pH8.7 balance it.

B. with protein example to 25mM Tris, 10mM NaCl, pH8.7 dialysis is washed post with same damping fluid behind the upper prop.

C. with the 10-150mM NaCl wash-out post that is added among the 25mM Tris (pH8.7).

4.a. collect the part that contains the 45KD product, merge the back to 25mM Tris, 10mMNaCl, the pH8.7 dialysis is concentrated into 1ml then in Speed Vac thickener.

B. enriched material is added on and uses 25mM Tris, 150mM NaCl is on Superdex 75 posts that the pH8.7 balance is crossed.As this product of homogeneous protein wash-out.Use the protein and the visible single band as shown in Figure 1B second hurdle of SDS-PAGE monitoring wash-out.F.32KD extracellular products (A)

1.a. discard 0-25% ammonium sulfate precipitation thing (0 ℃, 1 hour).

B. keep 25-60% ammonium sulfate precipitation thing (0 ℃ is spent the night).

2.a. with the 25mM Tris that contains 1M NaCl, pH8.7 filling DEAE CL-6B post is used 25mM Tris then, 10mM NaCl, the pH8.7 balance it.

B. with protein example to 25mM Tris, 10mM NaCl, pH8.7 dialysis is washed post with same damping fluid behind the upper prop and is spent the night.

C. with being added in 25mM Tris, salt gradient (10mM to 200mM) the wash-out post in the pH8.7 damping fluid.At the about wash-out 32KD of 70mM NaCl place protein.

3.a. collect the part contain the 32KD product, merge and to 25mM Tris, 10mMNaCl, the pH8.7 dialysis is concentrated to 1ml with protein example then in the Speed-Vac thickener.

B. make the gained enriched material by using 25mM Tris then, 150mM NaCl, Superdex 75 posts that the pH8.7 balance is crossed, and use this buffer solution elution.As homogeneous protein wash-out 32KD product.

4.a. with the 25mM Tris that contains 1M NaCl, pH8.7 loads Q-SepharoseHP post (Pharmacia), and uses 25mM Tris once more, 10mM NaCl, the pH8.7 balance it.

B. with protein example to 25mM Tris, 10mM NaCl, pH8.7 dialysis and upper prop are washed post with same damping fluid then.

C. use 100-300mM NaCl gradient elution post.At the 32A of about 120mM NaCl place wash-out, and provide as Figure 1B the single band shown in the hurdle 4 as the proteinic mark of homogeneous.G.32KD extracellular products (B)

1.a. discard 0-25% ammonium sulfate precipitation part (0 ℃ 1 hour).

B. keep 25-60% ammonium sulfate precipitation part (0 ℃ is spent the night).

2.a. with the 25mM Tris that contains 1M NaCl, pH8.7 loads DEAE CL-6B post (Pharmacia), uses 25mM Tris then, 10mM NaCl, the pH8.7 balance it.

C. cross and be added in 25mM Tris, first salt gradient of 10mM to the 200mM NaCl among the pH8.7 is with the wash-out range protein.Behind the balance columns, cross second salt gradient (200 to 300mM NaCl).At the about wash-out 32KD of 225mM NaCl place protein.

3.a. will contain the 25mM Tris of 1M NaCl, pH8.7 charges in the Q-SepharoseHp post (Pharmacia), and uses 25mM Tris once more, 10mM NaCl, the pH8.7 balance it.

B. with protein example to 25mM Tris, 10mM NaCl, pH8.7 dialysis, upper prop is being washed post then in same damping fluid.

C. with the 200-300mM NaCl gradient elution post that is added in the same damping fluid.

4.a. collect the part that contains the 32KD product, merge the back to 25mM Tris, 10mMNaCl, the pH8.7 dialysis is concentrated to 1ml with protein example then in the Speed-Vac thickener.

B. then enriched material is crossed and used 25mM Tris, 150mM NaCl (pH8.7) equilibrated Superdex 75 posts, and use same buffer solution elution.Be labeled as the 32KD product of 32B as homogeneous protein wash-out, so that itself and the isolating 32KD protein of using method H are distinguished, this product shows the single band shown in Figure 1B hurdle 3 in gel electrophoresis.H.30KD extracellular products

1.a. discard 0-25% ammonium sulfate precipitation thing (0 ℃ 1 hour).

B. keep 25-60% ammonium sulfate precipitation thing (0 ℃ is spent the night).

2.a. with the 25mM Tris that contains 1M NaCl, pH8.7 inserts DEAE CL-6B post (Pharmacia), uses 25mM Tris then, 10mM NaCl, pH8.7 balance.

C. with salt gradient (10mM to the 200mM) wash-out that is added in 25mM Tris (pH8.7) damping fluid.At the about wash-out 30KD of 140mM NaCl place protein.

3.a. collect the part that contains the 30KD product, merge the back to 25mM Tris, 10mMNaCl, (pH8.7) dialysis is concentrated to 1ml with protein example then in the Speed-Vac thickener.

B. then enriched material is splined on and uses 25mM Tris, also use this buffer solution elution on Superdex 75 posts that 150mM NaCl (pH8.7) balance is crossed., and be shown as the single band shown on the hurdle 5 as homogeneous protein wash-out 30KD product as Figure 1B.I.24KD extracellular products

1.a. discard 0-25% ammonium sulfate precipitation thing (0 ℃, 1 hour).

B. keep 25-60% ammonium sulfate precipitation thing (0 ℃ is spent the night).

2.a. will contain the 25mM Tris of 1M NaCl, (pH8.7) insert DEAE CL-6B post (Pharmacia), use 25mM Tris then, 10mM NaCl, (pH8.7) balance.

B. with protein example to 25mM Tris, 10mM NaCl, (pH8.7) dialysis, upper prop is also washed post with same damping fluid and is spent the night.

C. make be added in 10mM to the 200mM NaCl among the 25mM Tris (pH8.7) first salt gradient by post with the wash-out range protein.Add second salt gradient (200 to 300mM NaCl) behind the balance columns.At the about wash-out 24KD of 250mM NaCl place protein.

Be added in the Q-SepharoseHp post (Pharmacia) 3.a. will contain the 25mM Tris (pH8.7) of 1M NaCl, and use 25mM Tris, 10mM NaCl (pH8.7) balance it.

B. with protein example to 25mM Tris, 10mM NaCl (pH8.7) dialysis is washed post with same damping fluid behind the upper prop.

4.a. collect the part that contains the 24KD protein, merge the back to 25mM Tris, 10mM NaCl (pH8.7) dialysis is concentrated to 1ml with protein example then in the Speed-Vac thickener.

B. with the enriched material application of sample in using 25mM Tris, on the post that 150mM NaCl (pH8.7) balance is crossed and use same buffer solution elution., and be shown as the single band shown on the hurdle 7 as homogeneous protein wash-out 24KD product as Figure 1B.J.23.5KD extracellular products

1.a. discard 0-25% ammonium sulfate precipitation thing (0 ℃ 1 hour).

B. keep 25-60% ammonium sulfate precipitation thing (0 ℃ is spent the night).

Charge on the DEAE CL-6B post (Pharmacia) 2.a. will contain the 25mM Tris (pH8.7) of 1M NaCl, use 25mM Tris then, 10mM NaCl (pH8.7) balance columns.

B. with protein example to 25mM Tris, 10mM NaCl (pH8.7) dialysis is washed post with same damping fluid behind the upper prop and is spent the night.

C. with salt gradient (10mM to 200mM) the wash-out post that is added in 25mM Tris (pH8.7) damping fluid.At the about wash-out 23.5KD of 80mM NaCl place protein.

Charge into the Q-SepharoseHP post 3.a. will contain the 25mM Tris (pH8.7) of 1M NaCl, and use 25mM Tris once more, 10mM NaCl (pH8.7) balance it.

C. with the 100-300mM NaCl wash-out post that is added among the 25mM Tris (pH8.7).

D. repeating step 3a to 3c.

4.a. collect the part contain the 23.5KD product, merge and to 25mM Tris, 10mMNaCl (pH8.7) dialysis is concentrated to 1ml with protein example then in the Speed-Vac thickener.

B. then enriched material is splined on and uses 25mM Tris, on Superdex 75 posts that 150mM NaCl (pH8.7) balance is crossed, and use same buffer solution elution.As homogeneous protein wash-out 23.5KD product.Use the protein of SDS-PAGE monitoring wash-out, and produce as Figure 1B the single band shown in the hurdle 6.K.23KD extracellular products

1.a. discard the sedimentary part of 0-25% ammonium sulfate (0 ℃ hour) and 25-60% ammonium sulfate (0 ℃ is spent the night).

B. keep 60-95% ammonium sulfate precipitation thing.

Be added on the DEAE CL-6B post (Pharmacia) 2.a. will contain the 50mM Bis-Tris (pH7.0) of 1M NaCl, and use 50mM Bis-Tris, 100mM NaCl (pH7.0) balance it.

B. protein example is dialysed and upper prop to 50mM Bis-Tris (pH7.0) damping fluid that is added with 100mM NaCl, wash post with same damping fluid then and spend the night.

C. with 100 to the 300mM NaCl linear gradient elution posts that are added among the 50mM Bis-Tris (pH7.0).

D. collect the proteinic part of the 23KD that contains big 100-150mM NaCl place wash-out.

3.a. it is protein portion is saturating and be concentrated to 1-2ml on Savant Speed Vac thickener to 25mM Tris (pH8.7), 10mM NaCl.

B. enriched material is splined on and uses 25mM Tris, on Superdex 75 posts that 150mM NaCl (pH8.7), balance are crossed.As homogeneous protein wash-out such as Figure 1B, the protein shown in the hurdle 8.L.16KD extracellular products

1.a. discard 0-25% ammonium sulfate precipitation thing (0 ℃, 1 hour).

B. keep 25-60% sodium sulfate throw out (0 ℃ is spent the night).

Charge in the EDAE CL-6B post (Pharmacia) 2.a. will contain the 2.5mM Tris (pH8.7) of 1M NaCl, use 25mM Tris then, 10mM NaCl (pH8.7) balance it.

B. with protein example to containing the 25mM Tris of 10mM NaCl, (pH8.7) dialysis and upper prop, wash post with same damping fluid then and cross liquid.

C. with being added in 25mM Tris, (pH8.7) salt gradient (10mM to 200mM) the wash-out post in the damping fluid.At the about wash-out 16KD of 50mM NaCl place protein.

3.a. collect the part that contains the 16KD product, merge the back to 25mM Tris, 10mMNaCl (pH8.7) dialysis is arrived 1m1 with proteinic sample concentration then in the Speed-vac thickener.

B. then enriched material is splined on and uses 25mM Tris, on Superdex 75 posts that 150mM NaCl (pH8.7) balance is crossed, and with same buffer solution elution it.As homogeneous protein wash-out 16KD product.Use the protein of SDS-PAGE monitoring wash-out and form as Figure 1B the single band shown in the hurdle 9.M.14KD extracellular products

1.a. discard 0-25% ammonium sulfate precipitation thing (0 ℃, 1 hour).

B. keep 25-60% sodium sulfate throw out (0 ℃ is spent the night).

Be filled on the EDAE CL-6B post (Pharmacia) 2.a. will contain the 25mM Tris (pH8.7) of 1M NaCl, use 25mM Tris then, 10mM NaCl (pH8.7) balance.

B. with protein example to 25mM Tris, 10mM NaCl (pH8.7) dialysis and upper prop are washed post then and are crossed liquid in same damping fluid.

C. with being added in 25mM Tris, (pH8.7) salt gradient (10mM to 200mM) the wash-out post in the damping fluid.At the about wash-out 14KD of 60mM NaCl place protein.

3.a., (pH8.7) fill the Q-SpeharoseHP post, and use 25mM NaCl (pH 8.7) balance again with the 25mM Tris that contains 1M NaCl.

B. with protein example to 25mM Tris, 10mM NaCl (pH8.7) dialysis and upper prop are washed post with same damping fluid then.

D. repeating step 3a to 3c.

3.a. collection also merges the part that contains the 14KD product, to 25mM Tris, 10mMNaCl (pH8.7) dialysis is arrived 1ml with proteinic sample concentration then in the Speed-Vac thickener.

B. then with the enriched material application of sample in using 25mM Tris, 150mM NaCl (pH8.

7) on Superdex 75 posts that balance is crossed, and use this buffer solution elution.As homogeneous protein wash-out 14KD product.Use the protein of SDS-PAGE monitoring wash-out and be rendered as C, the single band shown in the hurdle 2 as Fig. 1.N.12KD extracellular products

1. obtain 0-10% ammonium sulfate precipitation thing (4 ℃ are spent the night).

2. the dissolved throw out is splined on the S200 Sephacryl size fractionation separator column again, and wash-out 12KD protein molecule.

With the protein portion application of sample on DEAE-Sepharose CL-6B or QAE-Sepharose ion exchange column, and press preceding method NaCl gradient elution.Wash-out contains two kinds of proteinic parts of homogeneous that molecular weight is about 12KD at about 300-350mM NaCl place, and collects it.This protein labeling is 12A and 12B, and as Fig. 1 D, the biobelt purifying shown in the hurdle 2 it.

As what show for example in the SDS-PAGE figure of Fig. 1, use the step that describes in detail among the embodiment 2A-2N enriching extracellular protein substantially or mainly and be purified to uniformity with mycobacterium tuberculosis.More particularly, Fig. 1 diagram shows use SOS-PAGE four 12.5% acrylamide gels of giving an example that represent and that be labeled as 1A, 1B, 1C and 1D.Standard substance in the gel 1A-1C swimming lane 1 comprise that molecular weight is 66,45,36,29,24,20 and the protein of 14KD.Among the gel 1D, it is 68,45,31,29,20 and the protein of 14KD that the standard substance on the swimming lane 1 contain molecular weight.Containing separately, the swimming lane of the extracellular products of purifying shows to have the band that indivedual protein have been reported molecular weight basically.It should be noted that in gel 1D, visible 12KD protein is dual band on the swimming lane 2.Sequential analysis shows that below 12KD band (or 12B KD band) is equal to top 12KD band (or 12AKD band), and different is that it lacks preceding 3 N-terminal amino acid.

The further analytical results that these is mainly enriched extracellular products individually is provided among Fig. 2.More particularly Fig. 2 is the N-terminal sequence data of tabulating relatively and to obtain from the extracellular products of these purifying, shows that most of isolating product is different really.Protein 32A, 32B and 30 have 5 identical N-terminal amino acid, therefore, and in order fully to identify and to distinguish these products and must do further sequential analysis.Fig. 3 shows the N-terminal aminoacid sequence of extension of the secretory product of these three kinds of purifying.Different amino acid proves on the position 16,31 and 36, although these isolating proteinic molecular weight are similar, they differ from one another.

Except protein 30,32A and 32B, also detected other and mainly enriched the N-terminal aminoacid sequence of the extension of extracellular products, the primary structure data to be provided and to find out possible relation between these protein.Use technology known in the art to carrying out sequencing according to the cellular products of embodiment 2 described method purifying.Provide in the following table by the apparent molecular weight of whole protein and assert, and the single-letter abbreviation of the standard of use represent natural amino acid, be used for the N-terminal aminoacid sequence of the different lengths of definite every kind of extracellular products.For consistent, from left to right write the N-terminal sequence to the direction of C-terminal with N-terminal with the record rule of having set up.Below the not too sure position of the amino acid whose identification of determining, ten days horizontal line is arranged.The amino acid that specific position is arranged is not measure or indefinite, and this position in sequence is represented with short-term.At last, two amino acid separately is that expression is correctly formed and clearly do not identified as yet and may occupy this position in this sequence by any amino acid by an oblique line.

The protein N terminal amino acid sequence

5 10 15 20 25 30 35

12KD FDTRL MRLED EMKEG RYEVR AELPG VDPDK DVDTM

40 45

VRDGQ LTIKA ERT

————————————

5 10 15 20 25 30

14KD ADPRL QFTAT TLSGA PFDGA S/NLQGK PAVL W

5 10 15 20 25 30

16KD AYPIT GKLGS ELTMT DTVGQ VVLGW KV SDL

35 40 45

F/YKSTA VIPGY TV-EQ QI

————————————

5 10 15 20

23KD AETYL PDLDW DYGAL EPHIS GQ

5 10

23.5KD APKTY -EELK?GTD

————————————

5 10 15 20 25 30 35

24KD APYEN LMVPS PSMGR DIPVA FLAGG PHAVY LLDAF

40 45 50 55 60

N AGPD?VSNWV TA GNA M MTLA -KGIC/S

—————————

5 10 15 20 25 30 35

30KD FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG NNSPA

40

VYLLD

—————————

5 10 15 20 25 30 35

32AKD FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG ANSP-

40

LYLLD

—————————

5 10 15 20

32B?KD FSRPG LPVEY LQVPS A-MGR DI

5 10 15 20 25 30

45KD DPEPA P PVP D? DAASP P DDAA APPA P ADPP-

—————————

5 10 15 20

58KD TEKTP DDVFK LAKDE KVLYL

—————————

5

71KD ARAVG I

—————————

5

80KD TDRVS VGN

—————————

5 10 15 20 110KD NSKSV NSFGA HDTLK V- ERK? RO

————————

This sequence data is used in combination the physical properties that SDS-PAGE determines again, can be identified and distinguish these representational extracellular products that mainly enrich of the present invention.Described analytical results shows, these protein have constituted and comprise 71KD, 30KD, 32AKD, 23KD and 16KD product, account for the most mycobacterium tuberculosis extracellular products of total extracellular products 60% (weight) that can utilize.Further estimate, 30KD protein may constitute by at the most 25% of the gross product weight of mycobacterium tuberculosis release.Therefore, the mycobacterium tuberculosis that in practice of the present invention, uses mainly enrich extracellular products individually can be from about 0.5% to about 25% of extracellular products gross weight.

As discussed above, can not identify the specific extracellular products of tool immunogen consistently owing to recognize traditional Western engram analysis method, so inventor's decision is analyzed the immunogenicity of mainly enriching extracellular products based on their abundance, and and then be easy to identify and separate it.The result is surprisingly found out that these mainly enrich extracellular products and have induced unpredictalbe effective immune response, the conclusion that causes the contriver to draw them to have the vaccine function.This wonderful discovery has caused the development of non-limiting functional theories of the present invention as discussed above.

In order to prove efficient of the present invention, use the single extracellular products that mainly enriches of various various dose to add experiment, to induce protective immunity in the laboratory animal model body that adopts in routine with its combination.More particularly, use purifying single mainly enrich extracellular products continue after be subjected to inducing protective immunity in the cavy body of mycobacterium tuberculosis.Use different route of administration that the various combination that mainly enriches extracellular products of five kinds of purifying is similarly tested, the result shows that these protein can induce protective immunity.Particularly use 30KD to enrich that the 71KD extracellular products of extracellular products and purified form is the same induces protective immunity in the animal model of approval.The same with the main abundant extracellular products of giving an example individually, five kinds of combination-vaccines that mainly enrich extracellular products have also been given the provide protection that antagonism lethal quantity mycobacterium tuberculosis is attacked.The result of every research that these vaccines of the present invention are done is as described below.

In comprising all experiments of carrying out the attack of immunogen or aerosol with mycobacterium tuberculosis, all use male Hartley strain cavy (the Charles RiverBreeding Laboratories of SFF, North Wilmington is Massachusetts) as

animal model.Divide

2 or 3 to be housed in the Rotating Stainless Steel Cage and cavy food and the water that freely is near the mark animal.After entering animal facility, observe one week of cavy at least, and then begin every experiment, to guarantee animal health.

3%-25% indivedual that use to estimate to account for total extracellular products of common existence mainly enrich extracellular products and carry out initial experiment.These experimental results show that mainly enriching extracellular products can cause effective immune response.More particularly, isolating 30KD show all that with the 71KD extracellular products can be created in cavy contacts the cell-mediated immune response that watches for animals behind the lethal quantity mycobacterium tuberculosis.

Embodiment 3

30KD protein with purifying makes a skin test to detect the cavy of 30KD immunity Cell-mediated immune response

Carry out the test of skin supersensitivity and can induce detectable immune response by the extracellular products that enriches of purified form to illustrate.Use according to what embodiment 2 described method purifying and a tree name letter contained the total extracellular products of about 25% mycobacterium tuberculosis and mainly enrich mycobacterium tuberculosis 30KD secretory product immune guinea pig.At three independently in the experiment, every three weeks with the 100mg 30KD protein cavy immunity three times of purifying in fact that is added in the SAF adjuvant.Inject to control animals according to the damping fluid that will be added among the SAF with quadrat method.Three weeks were attacked cavy to carry out the test of skin supersensitivity with 30KD protein after the last immunity.

Shave off guinea pig back Mao Bingjing intradermal injection 0.1, the 1 and 10 μ g 30KD protein that come into operation, check the erythema (skin redness) and the scleroma that are produced after 24 hours, shown in the following tabulation of the result A.Data are used average measurement value ± standard error (SE) expression of traditional method with each group.ND represents not do the experiment of this special aspects of the present invention.

Table A

To the proteinic erythematous response of 30KD (mm) (mean value ± SE)

The cavy state n 0.1. μ g 1.0 μ g 10.0 μ g Experiment 1

Immune group 6 1.2 ± 0.5 3.9 ± 0.8 6.9 ± 1.0

Control group 5 ND ND 3.0 ± 0.9 experiments 2

Immune group 6 0.5 ± 0.5 5.4 ± 0.7 8.1 ± 0.6

Control group 30 ± 0 2.5 ± 0 1.7 ± 0.8 experiment 3

Immune group 6 ND 1.7 ± 1.1 6.2 ± 0.3

Control group 3 ND ND 2.0 ± 0.0

To the proteinic scleroma of 30KD reaction (mm) (mean value ± SE) The cavy state n 0.1. μ g 1.0 μ g 10.0 μ g Experiment 1

Immune group 60 ± 0 3.3 ± 0.3 5.6 ± 0.9

Control group 5 ND ND 1.6 ± 1.0 experiments 2

Immune group 60 ± 0 3.8 ± 0.7 4.9 ± 1.2

Control group 30 ± 0 0.8 ± 0.8 1.7 ± 0.8 experiment 3

Immune group 6 ND 1.1 ± 1.1 4.7 ± 0.4

Control group 3 ND 0 ± 00 ± 0

Remarkable erythema shown in Table A and scleroma confirmed, shows strong cell-mediated immune response with the cavy of 30KD secretory product immunity.On the contrary, control animals then shows very little reaction.

In order further to confirm the immunoreactivity of 30KD secretory product and to show it, attack animal through the cavy of immunity and with this protein with m tuberculosis infection is non-to infectivity applicability lungy.

Embodiment 4

Detect m tuberculosis infection cavy cell-mediated immunoreactive purifying it The 30KD protein test

To infect the required bacterium of cavy in order obtaining to be used for testing, at first mycobacterium tuberculosis to be incubated on the 7H11 agar and and to go down to posterity once to guarantee that they have virulence by Guinea pig lung.For this reason, with containing about 5 * 10 ⁴The aerosol that is added in the 10ml bacterial suspension in the 7H9 nutrient solution of individual bacterium/ml is attacked cavy.After the cavy morbidity, kill animals and excision comprise the lung of tangible mycobacterium tuberculosis damage.Every leaf lung is ground and on 7H11 agar, cultivated 7 days to 10 days.Scrape from plate and to get bacterium, dilute in containing the 7H9 nutrient solution of 10% glycerine, supersound process is obtaining single cell suspension, and with about 2 * 10 in water-bath ⁷The concentration of individual viable bacteria/cell is slow freezing in-70 ℃.Through the survival rate of melting bacterial suspension and the serial dilutions of cultivation suspension detects freezing cell on 7H11 agar.Just before attacking, melt a bottle bacterial cell and in the 7H9 nutrient solution, be diluted to required concentration.

Make cavy in the synthetic glass aerosol chamber of a particular design, contact the aerosol of slip-knot nuclear mycobacterium.Aerosol chamber's size is 14 * 13 * 24 inches, and the door that comprises two 6 inch diameters on opposite sides is to be used for introducing or emitting cavy.The aerosol inlet is positioned at the center of cell top cover.(Gast Mfg.Co., Benton Harbor is Michigan) in the 30lb/ inch for vacuum pump ²Speed air is delivered to aerosol spray tube unit (Mes Inc., Burbank Cali-fornia) and from the 10ml bacterial suspension produces aerosol.One 0.2 μ m respiration cycle filtering unit (Pall Biomedical Inc., Fajardo, Puerto Rico) is positioned at an end of cell. in order to the inside and outside pressure of equilibrator.Be security consideration, with all placing the cell in the laminar flow hood to carry out the aerosol attack.

Make the former aerosol of animal contact sick body 30 minutes, the bacterial suspension in the complete during this period vent gas day with fog.At every turn from containing about 5.0 * 10 ⁴The 10ml suspension of bacteria particles/ml produces aerosol.Previous research shows that infection has all taken place the not protected cavy that contacts this concentration bacterium.After aerosol infects, cavy is housed in places the laminar flow safety screen (AiroClean Engineering Inc., Edgernont is Pernnsylvania) in Nei the Rotating Stainless Steel Cage and observe signs of disease.At whole experimental session, cavy food and water that animal can freely be near the mark.

In this experiment, kill infected cavy and detect the spleen lymphocyte proliferation situation that in 30KD proteins react, occurs various concentration.More particularly, press Brie-man and Horwitz (J.Exp.Med.164:799-811; Classify this paper reference as) described method preparation and purifying splenic lymphocyte.To be added in the RPMI 1640 (GIBCOLaboratories that contain penicillin (100v/ml), Streptomycin sulphate (100 μ g/ml) and 10% Guang bovine serum (GIBCO), Grancl Island, New York) lymphocyte in is transferred to the final concentration of 107/ml, and in micro-scale testing hole (tissue culturing plate at the bottom of 96 hole circles; Falcon Labware, Oxnard, California) in the cumulative volume of 100ml, at 5%CO ₂Be incubated 2 days in 37 ℃ of purifying 30KD secretory products under-95% air and 100% humidity condition with different concns.Use not infected animal as negative control.When insulation finishes, adding 0.25 μ Ci in each hole ( ³H) (New England NaClear, Boston is Mas-sachusetts) and at 5%CO for thymus pyrimidine ₂Under-95% air and 100% humidity environment cell is continued at 37 ℃ and be incubated 2 hours.(Skatron Inc., Sterling Vinginia) wash each aperture, and make effluent pass through filter pad (Skatron) to use various product automated cell harvesting device.The filter bed in the discrete micro-scale testing of representative hole partly is placed in the scintillation vial, and adds 2ml EcoscintH liquid scintillation mixed solution (National Diagnostics, Manville, New Jersey).(Califotnia) the middle beta-particle that detects is launched for Beckman Instruments Inc., Fullerton at the β scintillometer.

The isolating 30KD secretory protein of 1 and 10 μ g/ml detected derive from infected and the tissue sample of infected cavy not.Monitor then sample mix ( ³H) ability of thymidine.Tabulate down and provided the result of these detections among the B.

Data are that disclosure purpose provides as stimulation index, and it is defined as: with antigen be incubated lymphocytic average ( ³H) thymidine mix/do not add antigen insulation lymphocytic average ( ³H) thymidine mixes.

Table B is to the proteinic stimulation index of 30KD

The cavy state n 0.1. μ g/ml 10.0 μ g/ml

6 2.2 ± 0.2 9.7 ± 4.6 of infection

Control group 6 1.5 ± 0.3 2.0 ± 0.8

As shown in Table B, the cell of infected animal shows the proteinic strong reaction to 30KD, promptly occurs tangible dose-dependently splenic lymphocyte increment in the reaction of this mainly being enriched secretory product.On the contrary, not infected animal then only shows lymphocyte increment seldom.Therefore, the 30KD secretory product can the significantly immune response of inducing cell mediation in the mammalian body of m tuberculosis infection.

The protectiveness aspect of vaccine in order to demonstrate the invention is with the 30KD protein immune guinea pig of purifying and make animal contact mycobacterium tuberculosis as follows.

Embodiment 5

Attack the cavy of 30KD protein immunity with the mycobacterium tuberculosis of aerosolization

As previously mentioned, with three weekly intervals with being added in 100 μ g 30KD secretory proteins among the SAF with animal immune three times.With the whole EP immunity of the 120 μ g control group cavy that is added among the SAF, or with being added in damping fluid in figure one adjuvant to carry out vacation immune.In three weeks after the last immunity, the method described in the embodiment 4 of pressing is attacked animal with the aerosolization mycobacterium tuberculosis.Monitor the survival rate of three treated animals, and diagram is shown in the table 4.Tabulate down to have provided among the C and attack the absolute mortality ratio of determining in 14 weeks of back.

Table C

The cavy state Survival number/under fire count The percentage survival rate

4/6 67% of 30KD immunity

3/6 50% of EP immunity

1/6 17% of false immunity

As shown in Figure 4: be protected and avoid death with the cavy of 30KD protein immunity three times.Cavy with the immunity of 30KD protein has 67% survival approximately, and has only the control group cavy survival of 17% false immunity.

The body weight of also having monitored immunized animal keeps situation (data not shown goes out), and further specifies the prevention ability of being produced the vaccine that mainly enriches extracellular products by the present invention's instruction by pathogenic bacteria of having mixed.As if though immunized animal keeps its body weight, the high mortality of false immune animal makes can not make diagram relatively between immunized animal and control animals.

After the conclusion that draws the body weight study on monitoring, kill the animal of survival and detect slip-knot nuclear mycobacterium in every animal right lung and the spleen.Animal is immersed in the 2% amphyl solution (National Laboratiories, Montvale, New Jersey), and aseptic excision lung and spleen.The number of cardinal principle primary surface damage in the visual inspection counting lung.In 10ml 7H9, prepare each organ homogenate with mortar and pestle and 90 order Norton Alunduin (Fisher), serial dilution tissue homogenate in 7H9, and drip size with the suspension of every 0.1ml and cultivate each diluent on the plate duplicating of 7H11 agar, to detect colony forming unit (CFU) number of tuberculosis branch pestle bacterium in right lung and the spleen.All plates all remain in the pattern insulation can cell, and at 5%CO ₂, under 95% air and 100% humidity condition in 37 ℃ of insulations 12 to 14 days.Use this method to detect test, the result represents with average colony forming unit (CFU) ± standard error (SE) and is shown in down among the tabulation D.

Table D

Average CFU ± SE

The cavy state n Right lung Spleen

4 3.4 ± 1.7 * 10 of 30 KD immunity ⁷7.7 ± 3.9 * 10 ⁶

1 1.8 * 10 of false immunity ⁸8.5 * 10 ⁷

Logarithmic difference 0.73 1.04

As show as shown in the D, limited the growth of mycobacterium tuberculosis in lung and the spleen with the immunity of 30KD secretory protein.Though be used for that the comparison destination data just obtains from the false immune animal of a survival, but the CFU in the animal lung of the 30KD protein immunity of four survivals is than low 0.7 logarithmic value of false immune animal of survival, and low 1 logarithmic value of false immune animal of CFU ratio survival in the spleen.Based on the CFU counting of former proof and the high correlation between mortality ratio, the animal of survival has less CFU number than promptly dead animal before carrying out the CFU analysis probably in lung and spleen.The reduction of this CFU number has proved scope of the present invention and operability once more clearly in the lung of immunized animal and the spleen.

Also tested another kind of mycobacterium tuberculosis and mainly enriched the latent effect of immunoprotection that extracellular products is the 71KD extracellular products, to prove its immune protection with its isolating form.

Embodiment 6

With the 71KD protein of purifying the cavy of whole EP preparation immunity is carried out the skin examination Test

In order to prove that 71KD protein causes the ability of effective immune response in animal body, use this isolating extracellular products that mainly enriches the cavy with the immunity of whole mycobacterium tuberculosis preparation to be made a skin test with skin supersensitivity test method(s).As mentioned above, whole EP will give the acquired immunity that animal infects with Killing Mycobacterium Tuberculosis, but level of response is than littler with vaccine of the present invention.

The whole EP preparation of pressing method described in the embodiment 1 preparation with 120 μ g is with twice of three weekly interval immune guinea pig.Preparation is used for the vaccine of immunization in incomplete Freund's adjuvant, and false immune animal is only accepted damping fluid to replace EP.Three weeks shaved off the hair of respectively organizing guinea pig back after the last immunization, and through the 71KD protein of intradermal injection 0.1,1.0 and 10 μ g to make a skin test.Use 10.0 μ g damping fluids in contrast, and the cumulative volume of all injections is 0.1ml.Measure the diameter of erythema and scleroma after 24 hours, the results are shown in down among the tabulation E.The result of each group test all represents with the standard error (SE) that average measurement value ± the use traditional method is tried to achieve.

Table E

To the erythematous response (mm) of 71KD (mean value ± SE)

The cavy state n 0.1 μ g 1.0 μ g 10.0 μ g

4 6.5 ± 0.7 11.9 ± 1.4 18.9 ± 2.2 of immunity

Control group 3 2.5 ± 1.4 5.0 ± 2.9 11.8 ± 2.1

To the scleroma of 71 KD reaction (mm) (mean value ± SE)

The cavy state n 0.1. μ g 1.0 μ g 10.0 μ g

4 3.6 ± 1.1 6.8 ± 1.1 18.9 ± 0.8 of immunity

Control group 3 0.7 ± 0.7 3.7 ± 0.9 7.8 ± 1.0

The reaction of immunized animal almost is separately with the twice of the cavy reaction of damping fluid attacks, and with the Low Response of the animal of whole EP attack seldom (said whole EP) be same as be used for the EP of immune animal) (data not shown goes out).

In order further to confirm that the 71KD of purifying mainly enriches the immune response of extracellular products trigger cell mediation, kill the cavy of whole EP immunity, and detect to the spleen lymphocyte proliferation situation in the various concentration 71KD proteins reacts.Use non-immune animal in contrast.According to the method for embodiment 4 with lymphocyte with 71KD protein or do not add 71KD protein insulation 2 days, detect then they mix ( ³H) ability of thymidine.

Data are represented with the stimulation index of calculating by method described in the embodiment 4.The results are shown in that this 71KD protein is attacked tabulated down among the F.

Table F

To the stimulation index of 71KD (mean value ± SE)

The cavy state n 00.1. μ g/ml 0.1 μ g/ml 1.0 μ g/ml

4 1.5 ± 0.1 2.3 ± 0.5 8.1 ± 2.2 of immunity

Control group 2 1.7 ± 0.6 1.6 ± 0.4 2.5 ± 0.6

To the stimulation index of ED (mean value ± SE)

The cavy state n 0.01. μ g/ml 0.1 μ g/ml 1.0 μ g/ml

4 1.5 ± 0.1 2.2 ± 0.3 5.3 ± 1.4 of immunity

Control group 2 1.4 ± 0.2 1.5 ± 0.2 1.2 ± 0.1

As show as shown in the F, the result who obtains in the stimulation index of lymphocyte proliferation assay and the test of skin supersensitivity is similar.71KD and whole EP test sample all demonstrate the reaction higher 2 or 3 times than control group, show that isolating 71KD mainly enriches the immune response that extracellular products can trigger cell mediation in the animal body of mycobacterium tuberculosis extract immunity.But what should emphasize once more is, the main abundant or basic extracellular products of purifying not and vaccine of the prior art or the relevant problem of whole preparation and does not synthesize and commercially produces the vaccine of the present invention that is better than prior art easilier.

More particularly, can not make whole preparation in enormous quantities by modern molecular biology technique at an easy rate.Commercially producing these any methods that contain the non-refining whole preparation of the outer product of all cells should comprise the Pathogenic organisms of a large amount of cultivation target pathogenic agent or closely related kind and gather in the crops resulting supernatant liquor.This production method is easy to be polluted by target pathogenic agent, toxic by-products or other parasitic factors.In addition, the many immunogenicity determinants in these preparations cause the toxicity immune response possibly in by responsive person's body of immune population.Use the whole preparation of these non-purified also can cause to be used at present effectively the most general tuerculoderma of tuberculosis screening and control.

On the contrary, can use the host of high yield conversion with high relatively security mass production vaccine of the present invention.Equally, can be identical, realize that easily standardized means is produced vaccine of the present invention in batches, this is just in time opposite with the production of the outer product of whole cell of variation range broad.Moreover, because it is quite little to be the number of the immunogenicity determinant of passing host immune system, so greatly reduce toxic reaction and the chance that causes general screening invalidate the test.

Embodiment 7

Purifying 71KD proteinaceous skin test to the cavy of 71KD protein immunity

Mainly enrich extracellular products produces cell-mediated immune response in the animal body of whole EP immunity after this immune response of mainly enriching extracellular products can inducing cell mediating in the animal body of 71KD immunity that promptly shows purified form at the isolating 71KD of proof.

With three weekly interval phases, inoculate twice of cavy with the 71KD protein that is added in 100 μ g purifying among the SAF.With the same program false immune control animals of the damping fluid that is added among the SAF.Three weeks were attacked two treated animals with the isolating 71KD protein of 1 and 10 μ g intracutaneous after the last immunity.Measure the erythema and the scleroma size that are produced after 24 hours, the results are shown in down among the tabulation G.

Table G

Erythematous response (mm) (M mean value SE) to 71KD

The cavy state n 0 μ g 1.0 μ g 10.0 μ g

Through 30 of immunity ± 0 6.5 ± 1.5 15.0 ± 1.5

Control group 30 ± 0 2.7 ± 1.3 6.7 ± 1.3

To the scleroma of 71KD reaction (mm) (mean value ± SE)

The cavy state n 0 μ g 1.0 μ g 10.0 μ g

Through 30 of immunity ± 0 3.0 ± 1.0 9.3 ± 0.3

Control group 30 ± 00 ± 0 1.3 ± 1.3

In immune animal, the degree that scleroma and erythema form proves that much larger than the control animals without immunity inoculation vaccine of the present invention has caused the proteinic cell-mediated immune response of strong anti-71KD.

In order further to confirm this ability of enriching extracellular products itself according to technological guide effective immune response of the present invention, carried out lymphocyte proliferation assay.Kill the animal of process immunity as shown in table G, and the method for determining in the use-case 4 is carried out the spleen lymphocyte proliferation test.Attack the tissue sample of the cavy and the control group cavy that derive from the immunity of 71KD protein with the isolating 71KD protein of 0.1,1 and 10 μ g/ml, and monitor they mix ( ³H) ability of thymidine.Press preceding method and calculate stimulation index.Under the results are shown in of these tests the tabulation H in.

Table H

To the stimulation index of 71KD (mean value ± SE) The cavy state n 0.1 μ g/ml 1.0 μ g/ml 10.0 μ g/ml Through 3 4.0 of immunity ± 1.3 5.6 ± 2.5 12.2 ± 5.1 control group 3 1.3 ± 0.3 1.3 ± 0.3 3.2 ± 1.5

The same with the test of skin supersensitivity, 71KD protein immune animal has shown that the 71KD albumen to purifying has than the higher reaction of the immune control animals of vacation.Though can expect that the consequence that exogenous protein brings, these results clearly illustrate that and mainly enrich the immunoreactive ability that extracellular products has the inducing cell mediation.

Confirm isolating mainly enrich extracellular protein and will induce effectively cell-mediated immune response after, carry out further experiment as described below, be cross reactivity with the reaction that confirms this anti-tubercle bacillus.

Embodiment 8

71KD protein challenge infection tuberculosis with purifying

The cavy of mycobacterium

The method described in the embodiment 4 of pressing is used the cavy of aerosolization m tuberculosis infection without immunity.Utilize the protein derivatives (PPD-CT68 of purifying; Connaught Laborato-ries Ltd.) as the cell-mediated immune response of the infected animal appearance of the assurance of positive control as the m tuberculosis infection indication.Be widely used in PPD in the Mantoux test that detects the tuberculosis contact generally with the preparation of ammonium sulfate fractional separation method, and include the mixture for the small protein matter of 10KD of molecular-weight average.The immune response of PPD is come down to and uses the immune response that partly causes by the isolating whole EP of method described in the embodiment 1 similar.

Infect three weeks of back, the purifying of giving an example with 0.1,1 and 10 μ g mainly enrich 71KD extracellular protein intradermal injection attack cavy.With infected animals not in contrast and similarly attack with isolating protein.Measure erythema and scleroma size after 24 hours, the results are shown in the following Table I.

Table I

To the erythematous response (mm) of 71KD (mean value ± SE)

The cavy state n 0.1 μ g 1.0 μ g 10.0 μ g

7 9.5 ± 1.7 13.4 ± 1.3 19.7 ± 1.3 of infection

Control group 6 2.3 ± 2.3 3.5 ± 2.2 7.8 ± 1.9

To the scleroma of 71KD reaction (mm) (mean value ± SE)

The cavy state n 0.1 μ g 1.0 μ g 10.0 μ g

7 5.3 ± 1.8 8.7 ± 1.6 13.4 ± 1.1 of infection

Control group 60 ± 0 0.8 ± 0.8 0 ± 0

As shown in Table I, in the infected animal body that mainly enriches the extracellular protein attack with purifying of the present invention strong immune response has appearred.Form than infection animal is not big 3 to 4 times with regard to erythema in this reaction, just scleroma forms than infection animal is not strong 10 times, thereby confirms that further this advantage 71KD extracellular protein can induce strong cell-mediated immune response in the animal body of m tuberculosis infection.

In order further to confirm to kill these results infected and not infected animal, and carry out lymphocyte proliferation assay according to the method for embodiment 4.The tissue sample that detects these two groups of cavys is to 0.1,1 71KD protein that separates with 10 μ g/ml and the cell proliferative response of PPD.By preceding method monitor these samples mix ( ³H) ability of thymidine the results are shown in down among the tabulation J.

Table J

To the stimulation index of 71KD (mean value ± SE) The cavy state n 0.1 μ g/ml 1.0 μ g/ml 10.0 μ g/ml3 2.4 ± 0.5 6.2 ± 1.8 29.1 ± 16.2 control group 3 1.1 ± 0.1 2.6 ± 0.8 18.2 ± 6.1 of infecting

To PPD stimulation index (mean value ± SE) The cavy state n 0.1 μ g/ml 1.0 μ g/ml 10.0 μ g/ml3 1.0 ± 0.1 4.0 ± 1.5 11.4 ± 3.4 control group 3 0.9 ± 0.2 0.9 ± 0.03 1.5 ± 0.3 of infecting

With the unanimity as a result of cutaneous sensibility test, table J shows that infected tissue is than much higher without the stimulation index of immune sample.More particularly, infected animal is high 2 times without the control animals that infects to the proteinic average peak stimulation index ratio of 71KD, PPD then than high 3 times without the control animals that infects, is shown that the extracellular protein vaccine that mainly enriches of the present invention induced strong cell-mediated immune response in the animal body of m tuberculosis infection.

Behind the main abundant protein of 71KD and this cross reactivity between mycobacterium tuberculosis of proof purifying, carry out next step experiment to prove that these samples that mainly enrich extracellular products by the method for the invention purifying can stimulate effective immune response.

Embodiment 9

Mycobacterium tuberculosis with aerosolization is attacked

The cavy of 71KD protein immunity

In order to prove immune protection main abundant or the outer protein vaccine of elementary cell, three weeks were mainly enriched 71KD protein with twice of cavy immunity with 100 μ g by embodiment 2 described method purifying at interval.Whole EP or damping fluid immunity control animals with 120 μ g embodiment 1.All use the SAF adjuvant during all animals of immunity.3 weeks after the last immunity make a skin test to the cavy with the immunity of 71KD protein with 10 μ g 71KD protein, to estimate whether to have developed cell-mediated immune response.Press the cavy of method described in the embodiment 4 then with aerosolization m tuberculosis infection control animals and the immunity of 71KD protein.Infect monitoring and weighing the weight of animals in back 6 months.

The compare the weight of animals of visible false immunity of curve shown in Figure 5 is reduced, and the animal of 71KD and whole EP immunity shows relative normal weight increase.The data of each group are all represented with mean body weight ± SE.The mortality curve of same animal is shown among Fig. 6.The absolute death of having reported the research among the K of tabulating down is flat.

Table K

The state of cavy Survival number/under fire count The percentage survival rate5/8 62.5% of 3/6 50%EP immunity of 71KD immunity

0/6 0% of false immunity

Weight loss curve and mortality ratio clearly illustrate that the extracellular protein that mainly enriches of the present invention has been given preventative immune response.The fact that finishes preceding 100% death without the animal of immunity during monitoring has further confirmed this point.

Embodiment 10

Attack 71KD protein with the aerosolization mycobacterium tuberculosis

The cavy of immunity

Carry out the result of similar experiment, and show that the outer protein of the elementary cell that comes into operation can cause protective immunological reaction in animal body with embodiment before confirming.In this experiment, once more with 3 weekly interval phases with 100 μ g 71KD extracellular proteins (being added among the SAF) with immune three times of cavy.The control group cavy carries out the vacation immunity with the damping fluid that is added among the SAF.3 weeks were attacked animal with the aerosolization mycobacterium tuberculosis after the last immunity, and surveyed body weight weekly, monitored for 13 weeks altogether.Calculate mean body weight ± SE of every group of 6 cavys, and diagram is shown among the figure F.This curve shows that false immune animal has been lost greatly during monitoring and weighs sb., and then keeps suitable constant body weight through the animal of immunity.Because weight loss or " consumption " are one of classical symptoms lungy.So these results show tubercule bacillus and have been subjected to the inhibition of vaccine of the present invention in intravital growth of immunized animal and propagation.

The extracellular products that enriches by the inoculation unpack format has caused protective immunity in cavy, carry out the interspecies immunity reactivity of next step experiment with proof vaccine of the present invention, and further confirms the validity and the applicability of guinea pig model.

Embodiment 11

71KD protein test PPD positive patients with purifying

Cell-mediated immunity

In order to estimate the immunoreactive cell-mediated composition of people to the main abundant protein of 71KD, according to the standard lymphopoiesis detection method described in the embodiment 4 above, research derives from the peripheral blood lymphocyte of the positive and PPD negative individuals of PPD to this proteinic proliferative response.Positive PPD or tuberculin reaction are well known in the art as the indication of former contacted mycobacterium tuberculosis.When two days and four days, detect proliferative response and corresponding ( ³H) thymidine mixes.The data of these researchs are shown among Fig. 8 A and the 8B.Fig. 8 A shows two days later to the proteinic reaction of the horizontal 71KD of various dose; Same reaction when Fig. 8 B shows four days.

Show as diagram among Fig. 8 A and the 8B, the average peak stimulation index of PPD positive individuals than PPD negative individuals to the high twice of the proteinic reaction of 71KD, doubly to the reaction Senior Three of PPD.In the PPD positive individuals, to existing linear mutual relationship between 71KD protein and peak value stimulation index to PPD, the strong cell-mediated reaction that proved the advantage of mycobacterium tuberculosis or the main human body internal stimulus that enriches the former contacted mycobacterium tuberculosis of extracellular products.These data conform to the reactive characteristics that sees cavy, and prove that further guinea pig model can be applicable to study other through infected Mammals.

Therefore, the same with the 30KD protein of discussing in the past, the anti-strong immunoreactive development of mainly enriching the 71KD extracellular products has proved wide region of the present invention, and the fact shows that the 71KD product also can the immune response of irritation cell mediation effectively in human body.

In addition, what should emphasize is that the present invention is not only limited to the extracellular products of mycobacterium tuberculosis and uses 71KD protein.Technology of the present invention is also applicable to any extracellular products that mainly enriches as proving among the embodiment.

Finish further research, to determine whether to use mainly enriching extracellular products and also can providing protective immunity of mycobacterium tuberculosis.Generally speaking, these researchs are the vaccines that utilize with various various dose, and carry out through intracutaneous or subcutaneous immune three times cavy with 3 or 4 weekly interval phases, wherein said vaccine contains the mycobacterium tuberculosis extracellular protein that is added in 5 kinds of purifying among the SAF of combination by different way.

The first kind of protein that is labeled as combination I that is used for immune programme for children makes up 71KD, 32A KD, 30KD, 23KD and the 16KD protein that comprises by method purifying described in the embodiment 2.Believe that this combination contains the total extracellular protein of normal presence in mycobacterium tuberculosis culture supernatant up to 60%.Indicate these protein of selecting to be used to make up I with asterisk among Fig. 2.Come into operation with adjuvant SAF through intradermal injection and to contain 100 μ g, 20 μ g or each proteinic combination I vaccine of 2 μ g.In similar experiment, use in addition and contain each proteinic combination I vaccine of 20 μ g.Use equal-volume SAF and damping fluid immunity negative control cavy with same program, and use whole cell outer protein formulation (be added in SAF) the immune negative control cavy of 120 μ g by embodiment 1 described method preparation.All volume injected all use damping fluid to be transferred to standard volume.

Embodiment 12

The cavy of combination I immunity is to using combination I epidemic disease

The reaction that seedling is attacked

Carry out the test of skin supersensitivity, whether to have developed detectable immune response behind the combination I mixture of determining animal product outside the inoculation elementary cell.Shave the extracellular protein of five kinds of purifying of mao back intradermal injection 1.0 μ g and the same combination of 10.0 μ g at guinea pig back.Use 10.0 μ g damping fluids in contrast and all injections all carry out with the cumulative volume of 0.1ml.Inject the diameter of measuring tuerculoderma position erythema and scleroma in back 24 hours.

Measuring result is shown in down among the tabulation L.Data are all with standard error (SE) expression of the average measurement value of each group ± determine with traditional method.ND represents not carry out this specific detection.

Table L

The cavy state Erythema (mm) (mean value ± SE)

n PD 1.0μg 10.0μg

Through 60 11.4 of immunity ± 4.6 17.4 ± 2.6

Control group 60 ND 6.0 ± 0.5

Scleroma (mm) (mean value ± SE)

n PD 1.0μg 10.0μg

Through 60 7.3 of immunity ± 0.8 11.6 ± 1.2

Control group 60 ND 4.2 ± 0.3

These data prove that clearly vaccinated animal has produced the strong cell-mediated immune response of anti-combination I extracellular protein.Erythema and scleroma observed value through immune cavy are almost compared according to treated animal big three times.

Embodiment 13

The combination I vaccine immunity protectiveness of antagonism aerosolization mycobacterium tuberculosis is analyzed

In three weeks after the last immunity, attack the cavy that is used for aforementioned supersensitivity test with aerosolization mycobacterium tuberculosis (Erdman bacterial strain), and survey weekly continuous 10 weeks of the weight of animals.Carrying out this aerosol by method described in the embodiment 4 attacks.Attack the animal of the outer product immunity of elementary cell of 6 usefulness 100 μ g combination I simultaneously with the aerosolization mycobacterium tuberculosis, and onesize group feminine gender and negative control treated animal.Three immunizations of positive controls are added on 120 μ g EP among the SAF.

Promptly dead animal and check its sign of tuberculosis damage substantially before finishing during the anatomic observation.Find at the end during the research that all animals all have such damage.

Through repeating variance analysis (ANOVA), analyze the difference between aerosol attack back immune group and control animals mean body weight.The survival rate difference of immune group and control group cavy after the accurate analysis of experiments of two tail Fisher is attacked.Data are mean body weight ± standard errors (SE) of every group of 6 cavys.

The results are shown among Fig. 9 of measured body weight weekly after the attack.With compare with the cavy of extracellular products immunity of combination, the weight of animals of false immunity has reduced 15.9% of its TBW.The body weight of negative control group is similar to the weight of animals of the extracellular protein immunity of share five kinds of purifying.Directly get the stdn body weight before the attack.Through duplicate detection ANOVA, significant difference is arranged, p＜0.0000001 between the animal of discovery combination I vaccine immunity and the animal of false immunity.

Attack ten weeks half of back and determine mortality ratio.All three false immune animals are all dead in three days between ten and ten weeks half after attack.Under tabulate and provided the mortality ratio monitoring result among the M.

Table M

The cavy state Survival number/under fire count The percentage survival rate

6/6 100% of combined immunization

5/6 83% of EP immunity

3/6 50% of false immunity

After the conclusion that draws the body weight study on monitoring, put to death the animal of survival through causing hypercapnia, and use the slip-knot nuclear mycobacterium number in embodiment 5 described every animal right lungs of methods detection and the spleen.Tabulating down to have provided among the N comprises three count results dead animal of last week of implementing, and numerical value is with average colony forming unit (CFU) ± standard error (SE) expression.

Table N

Average CFU ± SE The cavy state n Right lung Spleen6 8.9 ± 5.4 * 10 of false immunity ⁷1.3 ± 0.7 * 10 ⁷6 3.4 ± 1.7 * 10 of immunity ⁶1.8 ± 0.6 * 10 ⁶6 1.7 ± 0.7 * 10 of EP immunity ⁷5.0 ± 2.8 * 10 ⁶

Logarithmic difference in the animal of the protein purification immunity of use associating and the animal lung of false immunity between bacterial concentration is 1.4, and the logarithmic difference of spleen inner cell concentration is 0.9.To compare side by side through the necrotomy tissue of the immune animal tissue with the control animals of false immunity, the tuberculosis damage obviously reduces in visible the former lung of gross examination of skeletal muscle.Compare with animal with the positive control treated animal of the whole cell external preparation (EP) of embodiment 1 immunity with the combination I mixture immunity of the extracellular protein of purifying, cell counting is Duoed 0.7 logarithmic value than the latter in the former lung, and the bacterial count value is Duoed 0.5 logarithmic value than the latter in the spleen.

Embodiment 14

Intracutaneous and the subcutaneous low dosage combination I vaccine that comes into operation

The immanoprotection action analysis that is produced

Though the dosage that embodiment 13 confirms with every kind of protein (30+32A+16+23+71KD) 100 μ g; can produce immanoprotection action three times with combination I protein every 4 all intracutaneous injecting immune animals, but also need do other research with the proof low dosage proteinic immune protection of combination I of each 20 μ g or 2 μ g particularly.Press method described in the embodiment 13, with 3 weekly interval phases with combination I protein (30+32A+16+23+71KD) with SAF intradermal injection immunity cavy 4 times.Animal is all accepted 20 μ g or every kind of protein of 2 μ g when immune at every turn.Control animals utilizes preceding method to carry out the vacation immunity.In 3 weeks after the last immunity, attack animal and in 9 weeks, survey the weight of animals weekly with the aerosolization mycobacterium tuberculosis.When all animals through immunity all survive to the experiment end, and a false immune animal is dead before experiment finishes.The following result that shows is indicated, has both made dosage than low 5 times even 50 times of the dosage that uses among the embodiment 13, still can protect immunized animal to avoid suffering the attack of aerosolization mycobacterium tuberculosis, and intracutaneous and subcutaneous all be effective by way of administration.

Compare with the cavy with the combination I vaccine immunity of each 20 μ g of every kind of protein, the animal of false immunity is 12% (demarcating body weight) of 9 all experimental session weight loss TBWs before attacking.Compare with cavy with the combination I vaccine immunity of each 3 μ g of every kind of protein, the animal of false immunity lost its demarcation TBW 11%.Therefore, the cavy with low dosage combination I protein intradermal injection immunity may stop losing of body weight after attacked by tuberculosis mycomycete aerosol.

Equally, can resist and the relevant excessive splenomegaly of mycobacterium tuberculosis dip-dye spleen with the cavy of low dosage combination I protein intradermal injection immunity.As show as shown in the O, with the average spleen of animal of the combination I protein immunity of each 20 μ g of every kind of protein or the 2 μ g (mean value ± SE) that heavily is respectively 4.6 ± 1.29 and 4.0 ± 0.89, and the animal spleen of the false immunity average out to 1.96 ± 1.8g that weighs, or up to the former more than 2 times.

Table O

Spleen weight (g)

The state of cavy n Mean value ± SE

5 9.6 ± 1.8 of false immunity

(the 20 μ g) 6 4.6 ± 1.2 of immunity

(the 2 μ g) 6 4.0 ± 0.8 of immunity

Cavy with low dosage combination I protein intradermal immunization also has less mycobacterium tuberculosis CFU counting in its spleen.As show as shown in the P, in the spleen with the cavy of the combination I protein immunity of each 20 μ g or 2 μ g, the CFU counting on average lacks 0.6 and 0.4 logarithmic value than false immune animal respectively.

Table P

CFU in the spleen The cavy state n Mean value ± SE Logarithmic difference5 3.1 ± 2.3 * 10 of false immunity ⁶(the 20 μ g) 6 8.1 ± 2.4 * 10 of immunity ⁵0.6 (the 2 μ g) 6 1.2 ± 0.6 * 10 of immunity ⁶-0.4

In addition, the cavy with the subcutaneous immunity of combination I protein also can stop weight loss, the growth in spleen of splenomegaly and mycobacterium tuberculosis.In the experiment identical with embodiment 14 described persons, also with 3 weekly interval phases with 20 μ g combination I protein through subcutaneous rather than intradermal immunization cavy 4 times.These animals are almost with using 20 μ g combination I protein equally to exempt from attack through the animal of intradermal immunization.

Embodiment 15

The cavy of combination I and combination II immunity is to combination I

Reaction with combination II attack

Carry out additional studies and also can provide protective immunity to determine whether other combinations that mycobacterium tuberculosis mainly enriches the extracellular protein product.Research be utilize contain two kinds of combinations promptly make up I (71,32A, 30,23 and 16KD) and make up the vaccine immunity cavy of II (32A, 30,24,23 and 16KD).Believe the total extracellular protein of normal presence in mycobacterium tuberculosis culture supernatant that contains among the combination II up to 62%.With the combination worker of containing 100 μ g range proteins or II (being added among the SAF) immune animal (6 every group) 4 times (each 3 weeks at interval).By same program equal-volume SAF and the false immune negative control treated animal of damping fluid.

By method described in the embodiment 12 animal is carried out the test of skin delayed hypersensitivity, to determine whether animal has produced detectable immune response after vaccine inoculation.In reaction, produce 16.8 ± 1.3mm (erythema of size of mean value ± SE) and the scleroma of 12.8 ± 1.2mm size with the animal of combination II immunity to the tuerculoderma of combination II; And the scleroma of erythema and 0.3 ± 0.3mm size of 1.3 ± 0.8mm size only appears in the animal of false immunity in the reaction to combination II.As seen, the animal erythema comparison of combination II immunity is big 12 times according to treated animal, and it is bigger 40 times than control group to harden.By contrast, 21.3 ± 2.0mm erythema and 15.8 ± 0.1mm scleroma appear in the animal of combination I immunity in the reaction to combination I tuerculoderma.And 6.4 ± 0.8mm erythema and 2.6 ± 0.7mm scleroma only appear in the animal of false immunity in the reaction to combination I.As seen, the animal erythema of combination I immunity is bigger 3 times than control group, and it is bigger 6 times than control group to harden.Just make up II protein, with the difference of control group even also bigger than the difference of combination I protein and control group.

In same experiment, (every kind of each 20 μ g of protein) have also produced the strong skin hypersensitivity reaction to combination II with low dosage combination II protein.They have the erythema of 21.0 ± 2.0mm and the scleroma of 15.3 ± 0.9mm in the reaction to combination II, and the erythema of 1.3 ± 0.8mm as mentioned above and the scleroma of 0.3 ± 0.3mm then only appear in the animal of false immunity.As seen, with the animal of low dosage combination II protein immunity, its erythema is bigger 16 times than control group, and it is bigger 50 times than control group to harden, with control group difference even than also big with the animal of high dosage combination II protein immunity.

Embodiment 16

Combination I and the anti-aerosolization tuberculosis of II vaccine branch

The immunoprotection analysis of bacillus

In 3 weeks after the last immunity, by attacking the cavy be used for aforementioned supersensitivity test with aerosolization mycobacterium tuberculosis (Erdman bacterial strain) described in the embodiment 13, and survey weekly continuous 7 weeks of body weight.With the same among the embodiment 13, every group of 6 animals.In preceding 7 weeks after attack, the animal mean body weight of false immunity is lost 19.5g.On the contrary, with the animal of combination II vaccine (every kind of each 100 μ g of protein) immunity then weight increase 52.4g, and with the animal mean body weight increase 67.2g of low dosage (every kind of each 20 μ g of protein) combination II protein immunity.By contrast, the weight of animals with combination I immunity has increased 68g.As seen, and compare with the cavy of combination II (100 μ g) immunity, the animal TBW of false immunity has reduced by 11%.Compare with the cavy that makes up the II immunity with low dosage (20 μ g), the animal TBW of false immunity has reduced by 14%.With compare with the animal of combination I immunity, the animal TBW of false immunity has also reduced by 14%.

Embodiment 17

Cavy with combination III to VII immunity is same to using

The reaction that vaccine or its composition are attacked

Add experiment to prove that various mycobacterium tuberculosis mainly enrich the validity of the combination of extracellular products.In these experiments, with containing 2 kinds or the multiple combination-vaccine that mainly enriches extracellular products by embodiment 2 described method purifying through intradermal injection immunity Hart-ley type cavy.Detect through SDS-PAGE, identify the extracellular products of these purifying according to its apparent molecular weight.Mainly enrich the extracellular products immune guinea pig with following combination.

Combination Protein component

III 30+32A+32B+16+23

IV 30+32A

V 30+32B

VI 30+16

VII 30+23

VIII 30+71

IX 30+23.5

X 30+12

XI 30+24

XII 30+58

Every kind of combination-vaccine contains every kind of listed protein of 100 μ g.Adjust the volume of combination-vaccine, and be added in and carry out intradermal injection among the adjuvant SAF.As previously mentioned, with 3 weekly interval phases with cavy immunity 4 times.

Carry out the test of skin supersensitivity to determine whether animal has developed detectable immune response after with combination III to XII vaccine inoculation.Every group of 6 guinea pig backs are shaved hair and intradermal injection and are used for the extracellular products that these animal users of time institute of immunity have the purifying of same combination.For carrying out this attack, injection is with the protein of the amount combination of each 10 μ g.All injections all use the cumulative volume of 0.1ml to finish.Also with combination III to XII protein, and the amount that is 10 μ g with every kind of protein in each combination makes a skin test to the false immune control animals with the SAF immunity only.Injecting the straight warp of measuring tuerculoderma position erythema and scleroma in back 24 hours by method described in the embodiment 3.

Under the results are shown in of these measurements the tabulation Q in.Standard error (SE) expression that all data are determined with the average measurement value ± use traditional method of this group.

Table Q

The diameter of skin reaction (mm)

The vaccine combination The tuerculoderma combination Erythema Scleroma

III III 12.2±2.0 6.8±0.8

IV IV 9.9±0.5 6.3±0.2

V V 13.0±1.1 8.1±0.7

VI VI 19.2±1.2 12.4±0.5

VII VII 14.3±1.0 8.7±0.4

VIII VIII 18.9±1.1 12.6±0.8

IX IX 17.0±0.9 12.1±0.9

X X 19.3±1.4 13.6±1.2

XI XI 18.3±1.2 12.4±0.8

XII XII 17.7±0.9 14.0±1.2

False III 4.8 ± 0.9 2.0 ± 0.0

False IV 4.3 ± 1.1 2.0 ± 0.0

False V 5.0 ± 0.5 2.0 ± 0.0

False VI 4.5 ± 0.3 2.0 ± 0.0

False VII 4.5 ± 0.3 2.0 ± 0.0

False VIII 3.3 ± 0.3 2.3 ± 0.3

False IX 3.7 ± 0.3 2.0 ± 0.0

False X 3.7 ± 0.4 2.0 ± 0.0

False XI 3.7 ± 0.2 2.0 ± 0.0

False XII 3.8 ± 0.2 2.0 ± 0.0

These results clearly prove and have produced the outer proteinic strong cell-mediated immune response of the purifying cells that resists various combinations.With regard to all combinations, the cavy of immunity demonstrate erythema big be 2 times of control group at least, normally 3 times or bigger.In addition, for all combinations, the scleroma size of the cavy of immunity is 3 times of control group at least.

Embodiment 18

The anti-aerosolization tuberculosis of combination III-XII protein

The immune protective analysis of mycobacterium

In order to prove these preventive effects of the outer product of purifying cells of combination for example, in three weeks after the program of use embodiment 4 is carried out last immunity, attack with the cavy that makes up the immunity of III to XII protein with mycobacterium tuberculosis.

Consistent with the result who early obtains, all protected and avoided death the useful cavy that makes up III to XII immunity of back institute under attack.There are 2 (33%) death in the 4th week after attack in the animal of 6 false immunity, and is then all not dead with the animal of combination IV-XIII protein immunity by contrast, and with having only 1 (17%) death in 6 animals of combination III protein immunity.Attack 10 weeks of back, the animal of false immunity has 50% death, relatively the animal with combination IX and XII immunity does not have death (0%), with combination III, IV, V, VI, X and respectively organizing of XI immunity 1 death (17%) is arranged in 6 animals, with 5 having only 1 death (20%) in the animal of combination VIII immunity, and in the animal that makes up the VII immunity, 6 have only 2 death (33%).

The cavy of ptomatopsia death before viewing duration finishes, and check its sign of tuberculosis damage substantially.All animals all had damage when discovery finished in research.

After the conclusion that draws mortality ratio research, handle through hypercarbia and to kill the animal of survival and to use the method for embodiment 5 to check slip-knot nuclear mycobacterium number in every animal spleen.The result is shown in down the tabulation R together with the logarithm reduction value form that begins from false immune animal with average colony forming unit (CFU) number.The spleen CFU count value of an animal is O in each group of asterisk indication of CFU value back.For calculating purpose, see 0 counting as 10 ³CFU/ spleen or 3log.

Table R

CFU in the spleen Vaccine group (average log number) Logarithm reduction value from false immunityIII 5.99 .5IV 5.41 1.1V 6.27 .3VI＜5.80*＞.7VII＜5.61*＞.9VIII 6.47 .1IX＜5.85*＞.7X＜5.74*＞.8XI 5.93 .6XII 6.03 .5 vacations 6.53--

Average mycobacterium tuberculosis colony forming unit number hangs down the 0.5log value than the control animals of false immunity at least in the animal spleen with combination III, IV, VI, VII, IX, X, XI and XII immunity.Particularly proof combination VI and VII are particularly effective, approximately make colony forming unit mean number reduce by 10 times.With average colony forming unit (CFU) number in the animal spleen of combination V and VIII immunity more immune than vacation respectively control group low 0.3 and 0.1log value.According to this remarkable reduction of colony forming unit among the animal organ of technology immunity of the present invention, the operability of the present invention on immunoprotection is described again.

Embodiment 19

Cavy with the combination XIII immunity of three kinds of various dose

Reaction to combination XIII attack

In order further to limit operability of the present invention and scope, and prove the effectiveness of extracellular products of the purifying of other array modes, press preceding method with different vaccine inoculation dosage immune guinea pigs.Specifically, be decided to be combination XIII and comprise 30KD, 32 (A) KD and the proteinic other combination of 16KD 3 kinds with 50 μ g, 100 μ g and 200 μ g and mainly enrich the extracellular products immune animal.Same previous embodiments, with 3 weekly interval phases with each treated animal of combination XIII protein intradermal immunization 4 times that is added in the various dose among the SAF.

Carry out the test of skin supersensitivity to determine whether immunity back animal has produced detectable immune response.Shave the combination XIII protein that mao extracellular products of every kind of purifying of back intradermal injection contains 10.0 μ g to back part of animal.The cumulative volume of injection is 0.1ml.Control animals to false immunity also makes a skin test with the combination XIII of same dosage.Inject the straight warp of measuring erythema and scleroma on the tuerculoderma position in back 24 hours.

The result represents with the standard error (SE) of the average measurement value of each group ± determine by traditional method and is shown in down among the S that tabulates.

Table S

The diameter of skin reaction (mm)

The vaccine combination Vaccine dose Erythema Scleroma

XIII 50 17.8±1.3 13.2±1.0

XIII 100 11.2±0.9 7.3±0.4

XIII 200 10.0±0.7 7.0±0.4

False 0 5.7 ± 0.5 0.2 ± 0.2

These results clearly prove once more, have produced the cell-mediated immune response of strong anti-combination XIII in each the treated animal body with three kinds of various dose combination XIII immunity.Animal performance through immunity has the erythema bigger 2 to 3 times than control group.Be in all cases, the big at least 35 times scleroma of control animals of minimal reaction to have occurred having through the animal of immunity more significantly than performance.

Embodiment 20

The anti-aerosolization tuberculosis of the combination XIII of three kinds of various dose

The immunoprotection analysis of mycobacterium

In order further to prove the protective immunity function of the vaccine of the present invention of various dose, 3 weeks were attacked the cavys (6 every group) that are subjected to immunity that are used for aforementioned skin supersensitivity test after the last immunity with the aerosolization mycobacterium tuberculosis.Using the method that is described in detail among the embodiment 4 to carry out aerosol attacks.Attack the control animals of 12 false immunity simultaneously.

Measure the results are shown among Figure 10 and provided respectively and organizing respectively that cavy is protected and the situation that avoids weight loss of body weight after the attack weekly with the combination XIII immunity of three kinds of various dose.Make up the net increase that the immune animal of XIII (100 and 200 μ g) in fact shows body weight with higher dosage, use animal to show relatively little weight loss than low dosage combination XIII (50 μ g) immunity.On the contrary, the animal of false immunity after attack in several weeks body weight reduce 22% of TBW approximately, and mean body weight reduces 182g in 10 all viewing durations.

The U that tabulates has down shown that the animal and the percentage body weight of control animals that stand immunity change, wherein change value with attack mean body weight when finishing when deducting the commence firing mean body weight and with the gained result mean body weight during divided by the commence firing try to achieve.Similarly, percentage provide protection average percentage value of losing weight of then deducting control animals from the average percentage weight increase of immunized animal or minimizing value is tried to achieve.

Table U

Immunogen dosage % body weight changes % and avoids weight loss

Combination XIII 50-4% 18%

Combination XIII 100+7% 29%

Combination XIII 200+5% 27%

False immunity--22%-

Result shown in the table U shows, than compare the body weight (18%-29%) that the animal of false immunity has been lost a great deal of through the animal of immunity.Figure 10 provides during the monitoring of 10 weeks, each group is mapped the clean weight loss situation of each treated animal of graphic extension to false immune animal through immune animal at interval weekly.Because weight loss or " consumption " are one of typical symptom lungy, so these presentation of results tubercule bacilluss are in the intravital growth of animal of process immunity and the inhibition that propagation has been subjected to the combination-vaccine of the present invention of three kinds of various dose.

Embodiment 21

The anti-combination of combination XIV-XVIII vaccine XIX-XVIII attacks

The immunoprotection analysis

For further proof scope of the present invention with can in cavy, test these five kinds of additional combination-vaccines of combination XIV to XVIII by the wide region of the effective vaccine of the technology of the present invention preparation.Assert the extracellular products of these purifying according to the apparent molecular weight that records with the SDS-PAGE method, and provide the composed as follows of each combination-vaccine:

Combination Protein is formed

XIV 30、32A、16、32B、24、23、45

XV 30、32A、16、32B、24、23、45、23.5、12

XVI 30、32A、16、32B、24、23

XVII 30、32A、16、32B、24、71

XVIII 30、32A、32B

I 30、32A、16、23、71

Except using new combination-vaccine and suitable contrast, also used combination I in these experimentalists and technicians.With combination XIV or the XV of each 50 μ g of range protein, and combination I, XVI, XVII and the XVIII (all being added in the SAF adjuvant) of each 100 μ g of range protein are through the intradermal injection immunity cavy.Per injection is 3 weeks at interval, and immune animal is 4 times altogether.

Carry out the test of skin supersensitivity by preceding method after the vaccine inoculation, whether developed detectable immune response to determine animal.Shave hair and give intradermal injection the former extracellular protein of purifying that is used for the same combination of these animals of immunity to guinea pig back.The each attack injected the suitable combination-vaccine that contains 10 μ g range proteins.The cumulative volume of injection is 0.1ml.Also the animal with the false immunity of each combined protein confrontation of same dosage makes a skin test.Inject back 24 hours diameters by erythema and scleroma on the measurement of method described in the embodiment 3 tuerculoderma position.

The result of these measurements is with standard overdue (SE) expression of average measurement value ± determine by traditional method, and is shown in the following Table V.

Table V

The diameter of skin reaction (mm)

The vaccine combination The tuerculoderma combination Erythema Scleroma

XIV XIV 13.3±0.7 9.1±0.4

XV XV 10.4±0.4 6.5±0.4

XVI XVI 8.0±1.8 5.1±1.0

XVII XVII 9.4±0.9 6.1±1.1

XVIII XVIII 13.6±1.2 8.7±0.7

I I 10.0±0.3 6.7±0.2

False XIV 5.5 ± 1.6 0.4 ± 0.2

False XV 6.1 ± 0.5 0.4 ± 0.2

False XVI 4.6 ± 1.4 0.4 ± 0.2

False XVII 5.7 ± 1.2 0.2 ± 0.2

False XVIII 2.1 ± 1.1 0 ± 0

False I 6.0 ± 1.2 0.6 ± 0.2

These results clearly prove, have produced anti-combination XIV to XVIII through the animal of immunity, and the strong cell-mediated immune response of anti-combination I.The animal performance that stands immunity has the erythema that is about control animals erythema twice size.More obviously be in all cases, the big at least 10 times scleroma of false immune group animal of minimal reaction to occur having through the animal of immunity than a performance.

Embodiment 22

Combination XIV-XVIII and the anti-aerosolization tuberculosis of combination I branch

The immunoprotection analysis of bacillus

For the immunoreactivity of the combination-vaccine that further confirms embodiment 21 and prove that they are to infectivity resistant applicability lungy, three weeks were attacked the cavy through immunity that is used for aforementioned skin supersensitivity test with the aerosolization mycobacterium tuberculosis after the last immunity, and monitored its body weight by the method for embodiment 4.12 false immune control animals that are used for embodiment 20 are also similarly attacked.The diagram as a result of these attacks is shown among Figure 11 and directly is shown in down among the tabulation W.

Mean body weight when the mean body weight when attack finishing deducts the commence firing, and with the gained result mean body weight during divided by the commence firing, change to determine the percentage body weight.Similarly, from through deducting the average percentage of the control animals amount of losing weight the average percentage weight increase of animal of immunity or the reduction, with definite percentage maintenance effect.

Table W

Immunogen The % body weight changes % avoids weight loss

Combination XIV 3% 25%

Combination XV-4% 18%

Combination XVI-15% 7%

Combination XVII-11% 11%

Combination XVIII-12% 10%

Combination I-11% 11%

False-22%

As show as shown in the W, be protected with the cavy of each combination-vaccine immunity and avoid weight loss.The animal that is subjected to false immunity has been lost 22% of its total body weight approximately.On the contrary, the prophylactic effect of combination-vaccine then causes one of them test group weight increase, and other groups have then reduced the weight loss amount.Specifically, increased by 3%, then only reduced it with the immune animals of other combinations and amounted to 4% to 15% of body weight with the weight of animals that makes up the XIV immunity.

These the results are shown among Figure 11, and the clean weight increase of each treated animal or minimizing are with measured value mapping weekly after wherein attacking with regard to aerosolization.Significant difference on this statistics that stands between immune animal and false immune animal that shows among Figure 11 provides further evidence for using combination-vaccine of the present invention to produce the immunoprotection reaction.

Embodiment 23

Cavy with three kinds of different adjuvant immunities is intravital

Cell-mediated immunoreactivity

In order further to prove the broad applicability and the versatility of vaccine preparation of the present invention, use different adjuvants to carry out immunogenicity research.Specifically to use three kinds of different adjuvants be Syntex adjuvant prescription I (SAF), incomplete Freund's adjuvant (IFA) and the adjuvant that contains monophosphoryl lipid A (MPL) the 30KD protein that to prepare three kinds of different immunogens respectively be purifying, combination I (30,32A, 16,23,71) and make up XIII (30,32A, 16) protein.Known these adjuvants can strengthen the immunoreactivity of organism when using with immunogen.

Use the combination I and the XIII that contain each 100 μ g of every kind of protein that are added in three kinds of different adjuvant prescriptions respectively, and the 30KD protein of about 100 μ g purifying, through the intradermal injection immunity cavy.Be total to immune guinea pig three times with every kind of prescription, each three weeks at interval.

Carry out the test of skin supersensitivity after the immunity, whether developed detectable immune response to determine cavy.Guinea pig back is shaved hair and give intradermal injection former their the identical immunogen of immunity that is used for.During attack, contain the combination I of each 10 μ g of every kind of protein and the 30KD protein of XIII or 10 μ g purifying with the injection of the cumulative volume of 100ml.With the various immunogen preparations that contain identical adjuvant the false immune cavy that inoculates one of three kinds of adjuvants is made a skin test.Attack back 24 hours by the erythema at embodiment 3 described methods measurement tuerculoderma positions and the diameter of scleroma.

The results are shown in the following Table X of these measurements, the same with data previously discussed, these results are also with the average measurement value of each group ± press standard error (SE) expression that statistical method is determined.

Table X

The diameter of skin reaction (mm)

Vaccine Adjuvant Tuerculoderma reagent Erythema Scleroma

30 SAF 30 10.7±1.6 5.8±1.5

30 IFA 30 8.8±0.7 4.6±0.7

30 MPL 30 10.2±1.7 5.3±1.5

XIII SAF XIII 7.3±0.5 4.1±0.5

XIII IFA XIII 6.8±0.9 3.5±0.5

XIII MPL XIII 6.3±0.4 3.4±0.3

I SAF I 6.9±0.6 4.0±0.3

I IFA I 6.8±0.2 3.6±0.3

I MPL I 7.4±0.4 3.9±0.5

False SAF 30 0.7 ± 0.7 1.0 ± 0

False IFA 30 0 ± 00 ± 0

False MPL 30 0 ± 00 ± 0

False SAF XIII 1.0 ± 1.0 1.0 ± 0

False IFA XIII 0 ± 0 0.3 ± 0.3

False MPL XIII 0 ± 00 ± 0

False SAF I 4.7 ± 0.3 1.0 ± 0

False IFA I 2.0 ± 1.0 0.7 ± 0.3

False MPL I 1.0 ± 1.0 0.7 ± 0.3

Shown in the data that provide in the Table X, the extracellular products of combination-vaccine of the present invention and purifying has caused strong cell-mediated immunogenic response when preparing together with different adjuvants.In addition, for immunogen separately, each in three kinds of adjuvants all provides roughly the same immunogen reaction.In general, comparison approximately occurs according to big 7 to the 10 times erythema of treated animal through immune animal, the scleroma size is then compared according to treated animal big 4 to 6 times approximately.

Vaccine of the present invention is united use to cause the ability of strong immunogen reaction with different adjuvants, helps realizing the vaccine optimizing.That is to say, can be mainly based on stability, be free from side effects, low-cost and easily complementary standard such as storages select to be used for adjuvant by technology production effective vaccine preparation described herein.When can be under the relative initial condition widely used vaccine preparation of exploitation, these and some other not directly related with immune response stimulating standards are particularly importants.

Embodiment 24

Combination XIX-XXVIII antagonism combination XIX-XXVIII attacks

The immunoprotection analysis

The combination-vaccine of ten kinds of other modes of use promptly makes up XIX to XXVIII and produces immune response, with further proof wide region of the present invention.Except using these new combination-vaccines and suitable contrast, also use combination IV and XIII as positive control to cause the immune response of cavy.Assert the extracellular products of purifying according to the apparent molecular weight that records with the SDS-PAGE method, and provide the composed as follows of each combination-vaccine:

Combination Protein is formed

XIX 30，32A，23

XX 30，32A，23.5

XXI 30，32A，24

XXII 30，32A，71

XXIII 30，32A，16，23

XXIV 30，32A，16，23.5

XXV 30，32A，16，24

XXVI 30，32A，16，71

XXVII 30，32A，16，32B

XXVIII 30，32A，16，45

IV 30，32A

XIII 30，32A，16

Cavy immune 4 times altogether, be 3 weeks the interval of per injection.Each combination-vaccine that is used for immune animal is formed by every kind of protein of each the 100 μ g that is added in the SAF adjuvant, so that total volume injected of 0.1ml to be provided.

Use method described in the embodiment 3 to carry out the test of skin supersensitivity to determine whether animal has developed detectable immune response behind the combination-vaccine that inoculation is selected.Shave hair and give intradermal injection former their extracellular protein of same combination purifying of immunity that is used for to guinea pig back.The protein combination that is used to attack animal is made up of every kind of protein of each 10 μ g.Also use the holoprotein of respectively organizing of same dosage that the control animals through false immunity is made a skin test.It is described to press embodiment 3, injects the diameter of measuring tuerculoderma position erythema and scleroma in back 24 hours.

The gained measuring result is represented with the average measurement value ± standard error of each treated animal, and is shown in down among the tabulation Y.

Table Y

The diameter of skin reaction (mm)

The vaccine combination Combination is surveyed in the skin examination Erythema Scleroma

XIX XIX 8.5±0.6 3.9±0.3

XX XX 8.2±0.3 3.7±0.3

XXI XXI 11.1±1.1 4.5±0.4

XXII XXII 9.4±0.8 4.3±0.4

XXIII XXIII 8.3±1.1 3.0±0.3

XXIV XXIV 8.5±0.9 3.4±0.5

XXV XXV 7.9±0.5 3.2±0.4

XXVI XXVI 8.9±0.7 3.3±0.5

XXVII XXVII 7.2±1.0 2.8±0.5

XXVIII XXVIII 8.5±0.5 2.8±0.3

IV IV 9.0±0.9 4.1±0.3

XIII XIII 9.4±0.9 4.3±0.3

False XIX 4.0 ± 2.6 1.0 ± 0

False XX 1.3 ± 1.3 1.0 ± 0

False XXI 3.5 ± 1.0 1.3 ± 1.3

False XXII 1.3 ± 1.3 1.0 ± 1.0

False XXIII 0 ± 0 1.0 ± 0

False XXIV 0 ± 0 1.0 ± 0

False XXV 0 ± 0 1.0 ± 0

False XXVI 2.3 ± 2.3 2.0 ± 1.0

False XXVII 0 ± 0 1.0 ± 0

False XXVIII 2.0 ± 1.2 1.0 ± 0

False IV 2.8 ± 1.6 1.0 ± 0

False XIII 1.5 ± 1.5 1.0 ± 0

The result who provides among the table Y shows, has produced the strong cell-mediated immune response of anti-combination XIX to XXVIII when attacking with same immunogen.Identical with afore-mentioned, combination IV and XIII have also caused strong cell-mediated immune response.The erythema that cavy presented through immunity is the twice of the immune control animals of corresponding vacation at least, and proof has general comparison according to the big reaction more than four times of treated animal.Equally, be the twice of non-immune control animals at least through the immune scleroma that animal presented, and generally all big 3 to 4 times.In animal, produced the immunoprotection operability that stronger in fact immune response has illustrated combination-vaccine of the present invention once more according to technology immunity of the present invention.

Those skill in the art will appreciate that some other advantage of vaccine of the present invention and method.For example, since be with individual compound or through the combination of the highly purified molecule selected as the purpose vaccine, rather than use whole bacterium or its composition, so vaccine of the present invention is compared with attenuation of the prior art or dead vaccine, it seldom might cause toxic reaction.In addition, molecular vaccine of the present invention can not resemble the individuality that threatens immunocompromised host the viable bacteria.In fact, can be that therapeutic purpose are used for infected individuality with composition of the present invention, to stimulate the specific immune response of the disease-resistant former factor.

Extracellular products is mainly enriched in the selectivity use or its immunogen analogue also can stop the development of conditioning gonosome liquid reaction, thereby can reduce the pathogenic effects of bacterium in the cell.Because the protective immunity that produces by the inventive method is at unconjugated proteinic, so any conditioning reaction all will only cause engulfing or destroy and mainly enrich extracellular products, rather than acceleration comprises bacterial parasite.In addition, selectivity uses the extracellular products of purifying can reduce the possibility that produces certain untoward reaction, because this reaction can not utilize those based on widely used screening and the control techniques of host to the identification of immunogen agent.Different with vaccine of the prior art is, still can use by pathogenic agent and express, but the immunoreactivity molecule that is not included in the vaccine that makes by the inventive method carries out shaker test.Use such immunogenic determinant in those have contacted target pathogenic agent individual, initiation to allow to carry out the reaction of suitable detection.

Opposite with the attenuated bacteria and the bacterium composition of prior art vaccine, another advantage of the present invention is to be easy to obtain and be easy to utilize technology known in the art to isolate the extracellular products of purifying in a large number.Because for most of interested pathogenic agent, immunoreactivity product of the present invention all is natural being discharged in the extracellular environment medium, so simplified the program of removing cell internal contamination thing and cell debris.In addition owing to use advantage or main enrich extracellular products or its immunogen analogue stimulates required immune response, so in most of production systems general equal expression level and the culture concentration that can gather in the crops product that improves.Therefore, no matter use what mode of production, all can finish separation in enormous quantities at an easy rate by conventional biochemical methods such as chromatography or ultrafiltration to required product.Build-in attribute and the characterization of molecules that is used for the present invention's immunogenic determinant helps producing constant consistent, standardized, the high-quality combination thing that is suitable for high volume applications to a great extent.

In addition, use purifying molecule compound, also can relatively easily syntheticly in enormous quantities produce immunocompetence vaccine composition of the present invention based on the advantage of target pathogenic agent or the main immunogen character of enriching extracellular products.For example, can use recombinant DNA technology that interested extracellular products or other epidemic focus analogue are cloned in the avirulence host bacteria, and can gather in the crops it safely.Can use molecule clone technology known in the art, separate and be equivalent to extracellular products interested, its analogue or its segmental DNA, and express it by the high-expression vector that is inserted in host cell such as the intestinal bacteria.Technology for example is found in II R.Anon, Synthetic Vaccines 31-71 (1987), Tam et al, Incorporationof T and B Epitopes of the Circumsporozoite Protein in a ChemicallyDefined Synthetic Vaccine Against Malaria, 171 J.Exp.Med.299-306 (1990), and Stover et al, Protective Immunity Elicited by Recom-binant Bacille Calmette-Guerin (BCG) Expressing Outer Surface Pro-tein A (OspA) Lipoprotein:A Candidate Lyme Disease Vaccine, 178 J.Exp.Med 197-209 (1993).

Equally, can use common lab technology and automatic sequence analytical technology, with the outer protein of pure relatively form chemical synthetic cell in enormous quantities, its analogue, homologue or immunoreactive protein matter subunit.The antigenic determinant of extracellular products of peptide subunit or lower molecular weight analogue this production model is equivalent to to(for) structure are attractive especially.The technology of producing less protein subunit is known in the art, and is found in II R.Anon, Syn-thetic Vaccine 15-30 (1987); A.Streitwieser, Jr.Introduction to Or-ganic Chemistry 953-55 (3rd ed., 1985). the technology that can substitute can be referring to Gross et al, " Nonenzymatic cleavage of Peptide Bonds:The Methion-ine Residues in Bovine Pancreatic Ribonuclease ", 237; The Journal ofBiological Chemistry No.6 (1962); Mahoney, " Migh-Yield Cleavageof Tryptophanyl Peptide Bonds by O-Iodosobenzoic Acid ", 18, Bio-chemistry No.17 (1979), and Shoolnik et al., " Gowococcal Pili ", 159, Journal of Experimental Medicine (1984).Also can use other immunogen technology as using the antiidiotype or the directed molecular evolution of peptide, Nucleotide or other molecules such as stand-in molecule, to produce effectively, can produce the immunoreactivity compound of required preventative reaction.Utilize naked DNA to be found in Robinson as the prior art of vaccine, Pro-tection Against a Lethal Influenza Virus Challenge by Immunizationwith a Hemagglutinin-Expressing Plasmid DNA, 11 Vaccine 9 (1993); Ulmer et al., Heterologous Protection Against Influenza byInjection of DNA Encoding a Varal Protein, 259, Science (1993).In addition, Tao et al., Idiotype/Granuloeyte-Macrophage Colony-Stimulat-ing Factor Fusion Protein as a Vaccine for B-Ceo Lymphoma, 362Nature discloses the technology that merges strong immunogenic protein afterbody in (1993); Also have, Good et al., Human T-Cell Recognition of the Circumsporozoite Pro-tein of Plasmodium Faiciparum:Immunodominant T-Cell DomainsMap to the Polymorphic Regions of the Molecule, 85, Proc, Natl.A-cad, Sci.USA (1988) and Gao et al., Identification and Characterizationof T Halper Epitopes in the Nucleoprotein of Influenza A Virus, 143, among the The Journal of Inomunology No.9 (1989) the t cell epitope gene map has been described.

All showed homology because many bacteriums belong to,, neither limit the invention to Mycobacterium or specific kind wherein or serotype or independent antituberculotic vaccine so previous embodiment just illustrates rather than limits the scope of the invention and content.What also should emphasize once more is that the common existence of homology between the kind of DNA of microorganism and respective egg white matter makes vaccine of the present invention might induce the cross reactivity immunity.Can provide other kinds of the selected Pseudomonas of antagonism and the cross reactivity immunity that serotype is attacked because mainly enrich the immunodominance epi-position of extracellular products, so it will be recognized by those skilled in the art, can use the extracellular products of a kind or the vaccine that the development of immunogen analogue resists the infected by microbes of another kind.

For example, 90% to 100% homology is arranged between Mycobacterium bovis and mycobacterium tuberculosis, and the height cross reactivity is being arranged aspect the initiation immune response.Therefore, can provide the provide protection in various degree of antagonism m tuberculosis infection based on the vaccines that enriches extracellular products of Mycobacterium bovis or other mycobacteriums, and vice versa.Therefore, use the suitable height homologous immunogenic determinant that mainly enriches extracellular products, provide the immunoprophylaxis reaction of the several kinds of bacteriums of resisting same Pseudomonas should consider to be included in the scope of the present invention.

It is again emphasized that to be pointed out that, can use for putting into practice the immunogenicity determinant that the present invention selects, so as to causing effective immune response by many different forms.Therefore, the one or more immunogenic determinants that mainly enrich extracellular products from selection to host immune system that present are not very strict, can change it for helping its production or administration.For example, can use whole extracellular products or it to comprise that any immunostimulation of aforementioned peptide, protein subunit, immunogenicity analogue and homologue partly prepares vaccine of the present invention.As long as can cause effective immunoprophylaxis reaction, the less protein subunit that mainly enriches extracellular products and molecule analogue thereof includes within the scope of the invention.In addition, for example fused protein or the extracellular products modified by known molecular recombination technology are compatible with technology of the present invention on the whole for the recombinant protein product.Moreover, the analogue of the selected immunocompetence determinant that produces on the immunogen, for example the peptide and the Nucleotide of antiidiotypic antibody or use orthogenesis derives from technology also are included in the scope of the present invention.

Equally, the preparation of immunogen agent and be not limited to the solution that protein or its analogue are made in adjuvant to presenting of host immune system.For example, the immunogenic determinant that is derived from suitable extracellular protein can express on bacterium, phage, mycoplasma or the virus of avirulence not of the same race and modify with recombinant technology.In this case, can prepare complete live organism and be used to stimulate required reaction.On the contrary, when in hostile environment, carrying out extensive vaccine inoculation, may need the very stable prescription that does not add adjuvant or additive.In addition, when standing severe condition such as freeze-drying or oral administration or capsule parcel, the vaccine preparation should help keeping the stability and the immunoreactivity of activeconstituents.Therefore, the present invention includes in a large number according to the desired use of product and the different ingredients of the fixed immunogenic determinant that contains the purpose vaccine.

Those skill in the art will appreciate that, can utilize normal experiment to determine various pathogenic agent and host's vaccine dose.At present, it is believed that minimum suitable dose will be the 0.1 μ g order of magnitude, but for suitable system, 2.0 μ g, 20.0 μ g, 100 μ g dosage, even 1mg dosage may be optimum.Can use any routine immunization technology known in the art and the order suitable dosage that comes into operation.

Those skilled in the art can recognize that also the present invention can embody with other particular forms, and does not deviate from spirit of the present invention or its clou.Just disclose the embodiment of its illustrative in the specification sheets of the present invention, should belong in the scope of the invention its other changes of carrying out.Therefore, the present invention is not only limited to the particular that this paper describes in detail.Exactly, the present invention's claim that awaits the reply just clearly defines scope of the present invention and content.

Sequence table (1) general information: (i) applicant: (ii) denomination of invention: abundant extracellular products and its are produced and (iii) sequence number of using method: (iv) mailing address:

(A) addressee:

(B) street:

(C) city:

(D) state:

(E) country:

(F) postcode: (v) computer-readable form:

(A) media type: Floppy dish

(B) computer: IBM PC compatibility

(C) operating system:

(D) software: (vi) present application materials:

(A) application number:

(B) applying date:

(C) classification: (viii) lawyer/proxy's information:

(A) title:

(B) number of registration:

(C) reference number/reel number: (ix) telecommunication information:

(A) phone:

(B) fax: the information of (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:1:

Asn Ser Lys Val Ser

The information of 15 (2) SEQ ID NO:2: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:2:

Thr Asp Arg Val Ser

The information of 15 (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:3:

Ala Arg?Ala Val Gly

The information of 15 (2) SEQ ID NO:4: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity is molecule type (ii): protein is (iii) supposed: do not have (v) clip types: N-terminal (vi) source:

(A) organism: mycobacterium tuberculosis (xi) sequence description: SEQ ID NO:4:

Thr Glu Lys Thr Pro

The information of 15 (2) SEQ ID NO:5: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linear (xi) sequence description: SEQ ID NO:5:

Asp Pro Glu Pro Ala

The information of 15 (2) SEQ ID NO:6: (I) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity is sequence description (ii): SEQ ID NO:6

Phe Ser Arg Pro Gly

The information of 15 (2) SEQ ID NO:7: (i) sequence signature: (A) length: 5 amino acid (B) type: amino acid (D) topology: linear (xi) sequence description: SEQ ID NO:7:

Phe Ser Arg Pro Gly

The information of 15 (2) SEQ ID NO:8:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity

(xi) sequence description: SEQ ID NO:8:

Phe Ser Arg Pro Gly

The information of 15 (2) SEQ ID NO:9:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity

(xi) sequence description: SEQ ID NO:9:

Ala Pro Lys Glu Asn

The information of 15 (2) SEQ ID NO:10:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linearity

(xi) sequence description: SEQ ID NO:10

Ala Glu Thr Tyr Leu

The information of 15 (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linear (xi) sequence description: SEQ ID NO:11:

Ala?Glu Thr?Tyr Leu

The information of 15 (2) SEQ ID NO:12: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linear (xi) sequence description: SEQ ID NO:12:

Ala Tyr Pro Ile Thr

The information of 15 (2) SEQ ID NO:13: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(C) topology: linear (xi) sequence description: SEQ ID NO:13:

Ala Asp Pro?Arg Leu

The information of 15 (2) SEQ ID NO:14: (i) sequence signature:

(A) length: 5 amino acid

(B) type: amino acid

(D) topology: linear (xi) sequence description: SEQ ID NO:14:

Phe Asp Thr Arg Leu

The information of 15 (2) SEQ ID NO:15: (i) sequence signature:

(A) length: 40 amino acid

(B) type: amino acid

(D) topology: linear (xi) sequence description: SEQ ID NO:15:

Phe?Ser?Arg?Pro?Gly?Leu?Pro?Val?Glu?Tyr?Leu?Gln?Val?Pro?Ser?Pro

1 5 10 15

Ser?Met?Gly?Arg?Asp?Ile?Lys?Val?Gln?Phe?Gln?Ser?Gly?Gly?Asn?Asn

20 25 30

Ser?Pro?Ala?Val?Tyr?Leu?Leu?Asp

35 40

Claims

1. be used for starting in mammalian hosts immunoreactive, the vaccine inoculation agent of anti-Mycobacterium infectious agent, said vaccine inoculation agent comprises:

Be selected from the proteinic at least a extracellular products that mainly enriches of mycobacterium tuberculosis 110KD protein, 80KD protein, 71KD protein, 58KD protein, 45KD protein, 32AKD protein, 32BKD protein, 30KD protein, 24KD protein, 23.5KD protein, 23KD protein, 16KD protein, 14KD protein and 12KD.

2. the vaccine inoculation agent of claim 1, wherein said at least a main abundant extracellular products are that mycobacterium tuberculosis 110KD protein or its have the N-terminal aminoacid sequence

5 10 15 20

NSKSV NSFGA HDTLA V- ERK ROImmunoreactivity homologue or fragment, wherein from left to right for N-terminal to the carbonyl end direction.

3. the vaccine inoculation agent of claim 1, the wherein said at least a extracellular products that mainly enriches is that mycobacterium tuberculosis 80KD protein or its have the N-terminal aminoacid sequence

5

Immunoreactivity homologue or the fragment of TDRVS VGN wherein from left to right are that N-terminal is to the C-terminal direction.

4. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 71KD protein or have the N-terminal aminoacid sequence

5

Immunoreactivity homologue or the fragment of ARAVG I wherein from left to right are that N-terminal is to the C-terminal direction.

5. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 58KD protein or have the N-terminal aminoacid sequence

5 10 15 20

Immunoreactivity homologue or the fragment of TEKTP DDVFK LAKDE KVLYL wherein from left to right are that N-terminal is to the C-terminal direction.

6. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 45KD protein or have the N-terminal aminoacid sequence

5 10 15 20 25 30

FSRPG P PVP D DAASP P DDAA APPA PImmunoreactivity homologue or the fragment of ADPP-wherein from left to right are that N-terminal is to the C-terminal direction.

7. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 32B KD protein or have the N-terminal aminoacid sequence

5 10 15 20

Immunoreactivity homologue or the fragment of FSRPG LPVEY LQVPS A-MGR DI wherein from left to right are that N-terminal is to the C-terminal direction.

8. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 32A KD protein or have the N-terminal aminoacid sequence

5 10 15 20 25 30

FSRPG LRVEY LQVPS PSMGR DIKVQ FQSGG

35 40

Immunoreactivity homologue or the fragment of ANSP-LYLLD wherein from left to right are that N-terminal is to the C-terminal direction.

9. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 30KD protein or have the N-terminal aminoacid sequence

5 10 15 20 25 30

FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG

35 40

Immunoreactivity homologue or the fragment of NNSPA VYLLD wherein from left to right are that N-terminal is to the C-terminal direction.

10. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 24KD protein or have the N-terminal aminoacid sequence

5 10 15 20 25 30

APYEN LMVPS PSMGR DIPVA FVAGG PHANY

35 40 45 50 55 60

Immunoreactivity homologue or the fragment of LLDAF NAGPD VSNWV TAGNA MMTLA-KGIC/S wherein from left to right are that N-terminal is to the C-terminal direction.

11. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 23.5KD protein or have the N-terminal aminoacid sequence

5 10

Immunoreactivity homologue or the fragment of APKTY-EELK GTD wherein from left to right are that N-terminal is to the C-terminal direction.

12. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 23KD protein or have the N-terminal aminoacid sequence

5 10 15 20

Immunoreactivity homologue or the fragment of AETYL DPLDW KYGAL EPHIS CQ wherein from left to right are that N-terminal is to the C-terminal direction.

13. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 16KD protein or have the N-terminal aminoacid sequence

5 10 15 20 25

AYPIT GKLGS ELTMT DTVGQ VVLGW

30 35 40 45

KV SDL F/YKSTA VIPGY TImmunoreactivity homologue or the fragment of V-EQ QI wherein from left to right are that N-terminal is to the C-terminal direction.

14. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 14KD protein or have the N-terminal aminoacid sequence

5 10 15 20 25 30 ADPRL QFTAT TLSGA PFDGA S/NL QGK PAVL WImmunoreactivity homologue or fragment, wherein from left to right be that N-terminal is to the C-terminal direction.

15. the vaccine inoculation agent of claim 1, wherein said at least a mainly to enrich extracellular products be mycobacterium tuberculosis 12KD protein or have the N-terminal aminoacid sequence

5 10 15 20 25

FKTRL MRLED EMKEG RVEVR AELPG

30 35 40 45

Immunoreactivity homologue or the fragment of VDPDK DVDIM VRDGQ LTIKA ERT wherein from left to right are that N-terminal is to the C-terminal direction.

16. the vaccine inoculation agent of claim 1, wherein said is to be selected from the analogue that comprises acceptable salt on peptide, homologue, fused protein, glycosylation thing and the immunology thereof with at least a said at least a compound that mainly enriches extracellular products that is similar to said pathogenic agent.

17. the vaccine inoculation agent of claim 1, it further comprises adjunvant composition.

18. the product of claim 1, wherein said product is the combination-vaccine that is used for starting the effective immune response of anti-Mycobacterium infectious agent in the mammalian hosts body, and said combination-vaccine contains:

Be selected from the proteinic multiple extracellular products that mainly enriches of mycobacterium tuberculosis 110KD protein, 80KD protein, 71KD protein, 58KD protein, 45KD protein, 32A KD protein, 32B KD protein, 32KD protein, 24KD protein, 23.5KD protein, 23KD protein, 16KD protein, 14KD protein and 12KD.

19. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD protein, 24KD protein, 23KD protein and the proteinic mixture of 16KD.

20. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 32B KD protein, 30KD protein, 23KD protein and the proteinic mixture of 16KD.

21. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein and the proteinic mixture of 30KD.

22. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32B KD protein and the proteinic mixture of 30KD.

23. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 30KD protein and the proteinic mixture of 16KD.

24. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 30KD protein and the proteinic mixture of 23KD.

25. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 71KD protein and the proteinic mixture of 30KD.

26. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 30KD protein and the proteinic mixture of 23.5KD.

27. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 30KD protein and the proteinic mixture of 12KD.

28. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 30KD protein and the proteinic mixture of 24KD.

29. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 58KD protein and the proteinic mixture of 30KD.

30. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 45KD protein, 32A KD protein, 32B KD protein, 30KD protein, 24KD protein, 23KD protein, the proteinic mixture of 16KD.

31. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 45KD protein, 32A KD protein, 32B KD protein, 30KD protein, 24KD protein, 23.5KD protein, 23KD protein, 16KD protein and the proteinic mixture of 12KD.

32. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 32B KD protein, 30KD protein, 24KD protein 23KD protein and the proteinic mixture of 16KD.

33. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 71KD protein, 32A KD protein, 32B KD protein, 30KD protein, 24KD protein and the proteinic mixture of 16KD.

34. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 32B KD protein and the proteinic mixture of 30KD.

35. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD and the proteinic mixture of 16KD.

36. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD protein and the proteinic mixture of 23KD.

37. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD and the proteinic mixture of 23.5KD.

38. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD and the proteinic mixture of 24KD.

39. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD protein and the proteinic mixture of 71KD.

40. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD protein, 23KD protein and the proteinic mixture of 16KD.

41. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD protein, 23.5KD protein and the proteinic mixture of 16KD.

42. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 30KD protein, 24KD protein and the proteinic mixture of 16KD.

43. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 71KD protein, 32A KD protein, 30KD protein and the proteinic mixture of 16KD.

44. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 32A KD protein, 32B KD protein, 30KD protein and the proteinic mixture of 16KD.

45. the combination-vaccine of claim 18, wherein said combination-vaccine comprise mycobacterium tuberculosis 45KD protein, 32A KD protein, 30KD protein and the proteinic mixture of 16KD.